WO1999042615A1

WO1999042615A1 - Methods for determining amounts of nucleic acids

Info

Publication number: WO1999042615A1
Application number: PCT/US1999/003198
Authority: WO
Inventors: Rajesh Patel; Edwin F. Ullman
Original assignee: Dade Behring Inc.
Priority date: 1998-02-18
Filing date: 1999-02-16
Publication date: 1999-08-26
Also published as: EP0988400A1

Abstract

The present invention relates to a method for detecting a single stranded target polynucleotide. A combination is provided, which comprises (i) a medium suspected of containing said 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. The present method may be used in conjunction with a method for amplifying a target polynucleotide or a plurality of different nucleotides. Kits for carrying out the above methods are also disclosed. The method is particularly applicable to the amplification and detection of RNA.

Description

METHODS FOR DETERMINING AMOUNTS OF NUCLEIC ACIDS

BACKGROUND OF THE INVENTION 1. Field of the Invention.

Significant morbidity and mortality are associated with infectious diseases. More rapid and accurate diagnostic methods are required for better monitoring and treatment of disease. Molecular methods using DNA probes, nucleic acid hybridizations and in vitro amplification techniques are promising methods offering advantages to conventional methods used for patient diagnoses.

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. The availability of radioactive nucleoside triphosphates of high specific activity and the ³²P labeling of DNA with T4 polynucleotide kinase has made it possible to identify, isolate, and characterize various nucleic acid sequences of biological interest. 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. The application of 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-

paper, cellulose paper, diazotized paper, or a nylon membrane. After the target nucleic acid is fixed on the support, 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.

When very low concentrations must be detected, the above method is slow and labor intensive, and nonisotopic labels that are less readily detected than radiolabels are frequently not suitable. Recently, a method for the enzymatic amplification of specific segments of

DNA known as the polymerase chain reaction (PCR) method has been described. This in vitro amplification procedure is based on repeated cycles of denaturation, oligonucleotide primer annealing, and primer extension by thermophilic polymerase, resulting in the exponential increase in copies of the region flanked by the primers. The 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 fragment whose length is defined by the distance between the 5' ends of the oligonucleotide primers. Other methods for amplifying nucleic acids have also been developed. These methods include single primer amplification, ligase chain reaction (LCR), nucleic acid sequence based amplification (NASBA) and the Q-beta-replicase method. Regardless of the amplification used, the amplified product must be detected.

After amplification of a particular nucleic acid, 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-

support. 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.

Usually, 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. Commonly, 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 that have been utilized include the Taqman method used by Roche Molecular Diagnostics. In this approach 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. -4-

Each of the above methods has limitations. Where two probes are required, increasing the amount of target increases the signal up to a point and then the signal fall off (the "prozone" phenomenon). Methods that require a capturable ligand in the target cannot be used on non-amplified nucleic acids nor are all amplification methods capable of introducing a ligand into the amplified product. Fluorescence polarization changes on binding are small and the sensitivity is therefore limited. Taqman is subject to problems with emission form the quencher, which limits sensitivity; GenProbe's chemiluminescent probe requires reagent additions prior to detection. It is desirable to have a sensitive, simple method for amplifying and detecting nucleic acids preferably, in a homogeneous format. The method should minimize the number and complexity of steps and reagents. Also, it is desirable to know the concentration of the amplified product in the reaction medium.

2. Description of the Related Art.

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. When target binds to the portion of the complementary sequence not bound to the labeled oligonucleotide, 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.

Rapid, non-separation electrochemiluminescent DNA hybridization assays for PCR products using 3'-labeled oligonucleotide probes is described by Gudibande, et al., (1992) Molecular and Cellular Probes. 6: 495-503. A related disclosure is found in international patent application WO 9508644 A1 (950330). -5-

Marmaro, et al., (Meeting of the American Association of Clinical Chemists, San Diego, California, November 1994, Poster No. 54) discusses the design and use of fluorogenic probes in TaqMan, a homogeneous PCR assay.

A 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).

Kemp, et a]., (1990) Gene, 94:223-228, disclose simplified colorimetric analysis of polymerase chain reactions and detection of HIV sequences in AIDS patients.

German patent application DE 4234086-A1 (92.02.05) (Henco, et al.) 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 (Longiaru, et al.) 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. Sequence polymerization by polymerase chain reaction is described by Saiki, et a{., (1986) Science, 230: 1350-1354. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase is described by Saiki, et aj. , Science (1988) 239:487.

U.S. Patent Applications Serial Nos. 07/299,282 and 07/399,795, filed January 19, 1989, and August 29, 1989, respectively, now U.S. Patent No. 5,508,178, describe nucleic acid amplification using a single polynucleotide primer (ASPP). A method for introducing defined sequences at the 3'-end of a -6-

polynucleotide is described in U.S. Patent Application Serial No. 08/140,369, filed October 20, 1993, now U.S. Patent No. 5,679,512. The disclosures of these applications are incorporated herein by reference including the references listed therein in the sections entitled "Description of the Related Art."

SUMMARY OF THE INVENTION One embodiment of the present invention is a method for detecting a single stranded target polynucleotide. A combination is provided, 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 is provided 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-

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. 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. In this approach a combination is provided, 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.

Another aspect of the present invention is a method for amplifying and detecting a plurality of different. 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.

Another embodiment of the present invention is a kit for use in amplification and detection of a target polynucleotide. The kit 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-

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 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.

Another aspect of the present invention is a kit for use in an amplification and quantitation of a specific RNA. The kit 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. For each of the sets (I) the first probe has a sequence P1 that is capable of hybridizing with a sequence PP1 of the second probe, (II) 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, (III) the first probe is incapable of hybridizing to the PP2 or the single strand, (IV) 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.

BRIEF DESCRIPTION OF THE DRAWINGS 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-

DESCRIPTION OF THE SPECIFIC EMBODIMENTS 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-β-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. Preferably, the first probe and the second oligonucleotide probe are allowed to bind to each other prior to combining them with the other assay components. When the sample is combined with the first and

second probes, 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.

Preferably, at least the first oligonucleotide probe has a label capable of generating a signal. With increasing amounts of the target polynucleotide, 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. By contrast in the present invention the sites of binding are not contiguous and, therefore, it is not apparent that strand displacement would occur. In its broadest aspect 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.

Before proceeding further with a description of the specific embodiments of the present invention, a number of terms will be defined. 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-

microorganisms, e.g., bacteria, yeasts, viruses, viroids, molds, fungi, plants, animals, humans, and the like. 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:

Corvnebacteria

Corynebacterium diphtheria

Pneumococci Diplococcus pneumoniae

Streptococci

Streptococcus pyrogenes

Streptococcus salivarus

Staphylococci Staphylococcus aureus

Staphylococcus albus

Neisseria

Neisseria meningitidis

Neisseria gonorrhea Enterobacteriaciae

Escherichia coli

Aerobacter aerogenes The colliform

Klebsiella pneumoniae bacteria

Salmonella typhosa Salmonella choleraesuis The Salmonellae

Salmonella typhimurium

Shigella dysenteria

Shigella schmitzii

Shigella arabinotarda

The Shigellae

Shigella flexneri Shigella boydii Shigella sonnei Other enteric bacilli Proteus vulgaris -13-

Proteus mirabilis Proteus species Proteus morgani Pseudomonas aeruginosa Alcaligenes faecalis Vibrio cholerae

Hemophilus-Bordetella group Rhizopus oryzae

Hemophilus influenza, H. ducryi Rhizopus arrhizua Phycomycetes

Hemophilus hemophilus Rhizopus nigricans Hemophilus aegypticus Sporotrichum schenkii

Hemophilus parainfluenza Flonsecaea pedrosoi

Bordetella pertussis Fonsecacea compact

Pasteurellae Fonsecacea dermatidis

Pasteurella pestis Cladosporium carrionii Pasteurella tulareusis Phialophora verrucosa

Brucellae Aspergillus nidulans

Brucella melitensis Madurella mycetomi

Brucella abortus Madurella grisea

Brucella suis Allescheria boydii

Aerobic Spore-forming Bacilli Phialophora jeanselmei

Bacillus anthracis Microsporum gypseum

Bacillus subtilis Trichophyton mentagrophytes

Bacillus megatehum Keratinomyces ajelloi Bacillus cereus Microsporum canis

Anaerobic Spore-forming Bacilli Trichophyton rubrum

Clostridium botulinum Microsporum adouini

Closthdium tetani Viruses

Clostridium perfringens Adenoviruses Clostridium novyi Herpes Viruses

Clostridium septicum Herpes simplex

Clostridium histolyticum Varicella (Chicken pox)

Clostridium tertium Herpes Zoster (Shingles)

Clostridium bifermentans Virus B Clostridium sporogenes Cytomegalovirus

Mycobacteria Pox Viruses

Mycobacterium tuberculosis Variola (smallpox) hominis Mycobacterium bovis Vaccinia Mycobacterium avium Poxvirus bovis Mycobacterium leprae Paravaccinia Mycobacterium paratuberculosis Molluscum contagiosum -14-

Actinomvcetes (fungus-like bacteria) Picornaviruses

Actinomyces Isaeli Poliovirus

Actinomyces bovis Coxsackievirus

Actinomyces naeslundii Echovi ruses Nocardia asteroides Rhinoviruses

Nocardia brasiliensis Mvxoviruses

The Spirochetes Influenza (A, B, and C)

Treponema pallidum Spirillum minus Parainfluenza (1-4)

Treponema pertenue Streptobacillus Mumps Virus monoiliformis Newcastle Disease Virus

Treponema carateum Measles Virus

Borrelia recurrentis Rinderpest Virus

Leptospira icterohemorrhagiae Canine Distemper Virus

Leptospira canicola Respiratory Syncytial Virus Trvpanasomes Rubella Virus

Mvcoplasmas Arboviruses

Mycoplasma pneumoniae

Other pathogens Eastern Equine Eucephalitis Virus

Listeria monocytogenes Western Equine Eucephalitis Virus Erysipelothrix rhusiopathiae Sindbis Virus

Streptobacillus moniliformis Chikugunya Virus

Donvania granulomatis Semliki Forest Virus

Bartonella bacilliformis Mayora Virus

Rickettsiae (bacteria-like St. Louis Encephalitis Virus parasites)

Rickettsia prowazekii California Encephalitis Virus

Rickettsia mooseri Colorado Tick Fever Virus

Rickettsia rickettsii Yellow Fever Virus

Rickettsia conori Dengue Virus Rickettsia australis Reoviruses

Rickettsia sibiricus Reovirus Types 1-3 Retroviruses

Rickettsia akari Human Immunodeficiency Viruses (HIV) Rickettsia tsutsugamushi Human T-cell Lymphotrophic Virus I & II (HTLV)

Rickettsia burnetti Hepatitis Rickettsia quintana Hepatitis A Virus Chlamydia (unclassifiable parasites Hepatitis B Virus bacterial/viral) Hepatitis nonA-nonB Virus Chlamydia agents (naming uncertain) Tumor Viruses

Fungi Rauscher Leukemia Virus

Cryptococcus neoformans Gross Virus

Blastomyces dermatidis Maloney Leukemia Virus -15-

Hisoplasma capsulatum

Coccidioides immitis Human Papilloma Virus

Paracoccidioides brasiliensis Candida albicans Aspergillus fumigatus

Mucor corymbifer (Absidia corymbifera)

Also included are genes, such as hemoglobin gene for sickle-cell anemia, cystic fibrosis gene, oncogenes, cDNA, and the like. The polynucleotide analyte, where appropriate, 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.

For purposes of this invention, 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. Such treatments are well-known in the art and include, for instance, heat or alkali treatment. For example, 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. 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-

of the polynucleotide analyte flanked by the primers. 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.

Another method for achieving the result of an amplification of nucleic acids is known as the ligase chain reaction (LCR). This method uses a ligase enzyme to join pairs of preformed nucleic acid probes. The probes hybridize with each complementary strand of the nucleic acid analyte, if present, and ligase is employed to bind each pair of probes together resulting in two templates that can serve in the next cycle to reiterate the particular nucleic acid sequence.

Another method for achieving a nucleic acid amplification is the nucleic acid sequence based amplification (NASBA). This method is a promoter-directed, enzymatic process that induces in vitro continuous, homogeneous and isothermal amplification of a specific nucleic acid to provide RNA copies of the nucleic acid. 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-

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. Thus, 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. In 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.

In general, in 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. In general, 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-

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. Very roughly, 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. For short sequences (up to about 100 nucleotides) chemical synthesis will frequently be more economical as compared to the biological synthesis. In addition to economy, chemical synthesis provides a convenient way of incorporating low molecular weight compounds and/or modified bases during the synthesis step. Furthermore, 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. For longer sequences 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-

Other methods of 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. Oligonucleotide primer(s) -- an oligonucleotide that is usually employed in a chain extension on a polynucleotide template such as in, for example, an amplification of a nucleic acid. 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. Normally, 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. Usually, 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. Examples of such derivatives or analogs, by way of illustration and not limitation, 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. Usually, the catalysts are enzymes, such as DNA polymerases, for example, prokaryotic DNA polymerase (I, II, or III), T4 DNA -21-

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.

Wholly or partially sequentially -- when the sample and various agents utilized in the present invention are combined other than concomitantly (simultaneously), 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 (hybridizing) 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. Complementary - Two sequences are complementary when the sequence of -22-

one can bind to the sequence of the other in an anti-parallel sense wherein the 3'- end of each sequence binds to the 5'-end of the other sequence and each A, T(U), G, and C of one sequence is then aligned with a T(U), A, C, and G, respectively, of the other sequence. 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 - a nucleotide polymerase or a single stranded template polynucleotide having a sequence other than at its 3'-end that can hybridize to at least the 3'-end of the primer or both. 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.

Member of a specific binding pair ("sbp member") - one of two different molecules, having an area on the surface or in a cavity which specifically binds to and is thereby defined as complementary with a particular spatial and polar organization of the other molecule. The members of the specific binding pair are -23-

referred to as ligand and receptor (antiligand). These 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. Ligand - any compound for which a receptor naturally exists or can be prepared.

Receptor ("antiligand") - 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.

Support or surface -- a porous or non-porous water insoluble material. 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. -24-

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. Usually 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. In general, 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. 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. As mentioned above, 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. Examples of particular 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.

Signal Producing System - 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.

Preferably, 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. For example, in one approach 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. In another approach the 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. In another approach the 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. -26-

Preferably, when the first member is brought into close proximity with the second members of the signal producing system, a signal is produced.

A number of signal producing systems in accordance with the above may be employed. The following discussion is by way of illustration and not limitation. In one such system 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. By employing different second enzymes signals are produced that can be differentiated and used to determine a ratio of signals that is related to concentration of the target polynucleotide.

A list of enzymes is found in U.S. Patent No. 4,299,916 at column 30 to column 33. As mentioned above, of particular interest in the subject invention is the use of 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-

enzyme. For example, 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. For enzyme labels, 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.

In another approach 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. As will be appreciated, when the quencher is brought into close proximity to the fluorescent molecule by the formation of a termolecular complex, the fluorescence of the medium is decreased because of the absorption by the quencher of the light emitted by the fluorescer. 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-

aminopyridinium salts, hellebrigenin, tetracycline, sterophenol, benzimidazolylphenylamine, 2-oxo-3-chromen, indole, xanthene, 7-hydroxycoumarin, 4,5-benzimidazoles, phenoxazine, salicylate, strophanthidin, porphyrins, triarylmethanes, flavin and rare earth chelates oxides and salts. Exemplary fluorescers are enumerated in U.S. Patent No. 4,318,707 at columns 7 and 8, the disclosure of which is incorporated herein by reference.

A diverse number of energy absorbers or quenchers may be employed. 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. Generally, the quencher is a fluorescent compound or fluorescer but energy acceptors that have weak or no fluorescence are also useful. For example, one group of 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. Other examples of 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.

Another example of 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. Besides 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. -29-

As mentioned above, 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. A further discussion of 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.

In another approach 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. Alternatively, 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. 5,340,716 at column 19, line 30, to column 20, line 45, and column 22, line 58, to column 30, line 10, the disclosure of which is incorporated herein by reference. 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. Preferably, these compounds react with singlet oxygen to form dioxetanes or dioxetanones. The latter are usually electron rich olefins. Exemplary -30-

of such olefins are 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. In the present invention 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. Ancillary Materials - Various ancillary materials will frequently be employed in the methods carried out in accordance with the present invention. For example, buffers will normally be present in the assay medium, as well as stabilizers for the assay medium and the assay components. Frequently, in addition to these additives, 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.

As mentioned above one aspect of the present invention provides for detection of a target polynucleotide and its products produced in a nucleic acid -31-

amplification reaction. The present invention has particular application to amplification reactions conducted at isothermal temperature. When an amplification is employed, 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. At the very least, 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.

In one embodiment of the present invention for detecting a single stranded target polynucleotide, a combination is provided, 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. In general, for S1 and S2, as well as for PP1 , PP2 and P1 , 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. Accordingly, 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. -32-

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. However, the degree of complementary of P 1 to PP1 , as well as that of S2 to PP2 and S1 to PP1 , depends on the relative lengths and nucleotide composition of P1 , PP1 , S2, PP2 and S1. It is preferred that S2 be greater than about 90% complementary, preferably, greater than about 95% complementary, most preferably, fully complementary, to PP2. . It is preferred also that 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. When 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. There may be less than a total of 30 unhybridized (with respect to a single polynucleotide strand to which S1 and S2 or PP1 and PP2 are hybridized) nucleotides, more preferably, 1 to 10 unhybridized nucleotides, immediately before or after the loop and prior to S1 and S2 hybridized contiguously to their complementary sequences. This will be illustrated in more detail below.

In carrying out an amplification as part of the present method, an aqueous medium is employed. In general, an aqueous medium is employed for the entire method in accordance with the present invention. Other polar cosolvents may also -33-

be employed, usually oxygenated organic solvents of from 1-6, more usually from 1-4, carbon atoms, including alcohols, ethers and the like. Usually these cosolvents, if used, are present in less than about 70 weight percent, more usually in less than about 30 weight percent. 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. In general for amplification, the pH and temperature are chosen based on the particular method of amplification employed.

For example, for amplification involving temperature cycling and primer extension such as in PCR or single primer amplification, 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. In conducting PCR amplification of nucleic acids, 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. When the present invention includes an amplification involving temperature cycling such as in PCR, the present method is preferably employed at the conclusion of the temperature cycling. -34-

For amplification by NASBA and 3SR, the reaction is conducted at 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 microμmolar or more, usually 10^"12to 10 ^"7M, more usually 10^"10to 10 ^"7M.

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 10¹⁰, more usually from about 10³ to 10⁸ molecules in a sample preferably at least 10^"21M in the sample and may be 10^"10 to 10^"19M, more usually 10^"14 to 10^"19M. For amplification by primer extension, 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 μL. Usually, the primer(s) are present in at -35-

least 10^"9 M, preferably 10^~7 M, and more preferably at least about 10^"6 M. Preferably, 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. For amplifications involving primer extension, the 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^"2M, preferably 10^"5 to 10^"3M.

After an amplification is carried out, or alternatively where no amplification is conducted, 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. Preferably, 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.

The presence of displaced first probe and/or the disappearance of a duplex comprising the first and second probe is then detected. To this end one or both of the 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, and the conditions therefor, 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. If the reporter molecule is a fluorescent molecule, the medium can be irradiated and the fluorescence determined. Where 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.

In one aspect of the present invention 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. Such a 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-

Preferably, 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, for example, 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.

Briefly, 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-

When the amplification uses a DNA polymerase, preferably, both of the oligonucleotide probes are blocked at the 3'-end to avoid any potential interference with and during amplification. To this end, 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. Alternatively, 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. For example, in order to introduce dextran at the 3'-end 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. Then, 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. As can be seen, 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. To determine the presence and/or -39-

amount of S in a sample, a determination can be made as to the presence of PP-S or free P. It is apparent from the embodiment depicted that 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.

When the method of the present invention is carried out in conjunction with an amplification, the target nucleotide S is present in the reaction medium. As 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.

Another embodiment in accordance with the present invention is depicted in Fig. 2 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. First oligonucleotide probe P' comprises sequence P'1 and 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. Then, 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. As can be seen, 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'. Accordingly, PP'1 and PP'2 are noncontiguous. As with the embodiment above, to determine the presence and/or amount of S' in a sample, a determination can be made as to the presence of PP'-S' or free P'. -40-

Another embodiment in accordance with the present invention is depicted in Fig. 3 by way of illustration and not limitation. In this embodiment an amplification of a target polynucleotide is carried out. 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. In the embodiment shown, 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. 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. As can be seen, 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-

hybridized to PP'. Accordingly, PP" 1 and PP"2 are noncontiguous. When the strand displacement of P" from PP" is complete, the signal is greatly diminished.

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 10⁷ or more copies per sample.

The nature of the signal from each of the reference polynucleotides and that for the target polynucleotide must be separately detectable. This entails iifferent signal producing system members for each of the above. For example, in the example depicted in Fig. 3, 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. Preferably, 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. In place of the target S2 sequence, 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. Less desirably, 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. Alternately, both the S1 and S2 sequences of the target are replaced by similar length arbitrary sequences in the reference. -42-

As mentioned above, 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.

As a matter of convenience, predetermined amounts of reagents employed in the present invention 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.

Another example of a kit in accordance with the present invention is a kit for RNA amplification. Such a kit 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. For each of the sets (I) the first probe has a sequence P1 that is capable of hybridizing with a sequence PP1 of the second probe, (II) 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, (III) the first probe is incapable -43-

of hybridizing to the PP2 or the single strand, (IV) 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. For example, in the case of NASBA, 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. In another example for PCR 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). The kit may also include various reference oligonucleotides and their respective signal producing system members as necessary.

The relative amounts of the various reagents in the kits can be varied widely to provide for concentrations of the reagents necessary to achieve the objects of the present invention. Under appropriate circumstances 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-

EXAMPLES The invention is demonstrated further by the following illustrative examples. Temperatures are in degrees centigrade (°C) and parts and percentages are by weight, unless otherwise indicated. Unless otherwise indicated, oligonucleotides used in the following examples were prepared by synthesis using an automated synthesizer and were purified by gel electrophoresis or HPLC.

The following abbreviations have the meanings set forth below:

Tris HCI - Tris(hydroxymethyl)aminomethane-HCI (a 10X solution) from BioWhittaker, Walkersville, MD.

DTT - dithiothreitol from Sigma Chemical Company, St. Louis, MO.

HPLC - high performance liquid chromatography.

DPP - 4,7-diphenylphenanthroline from Aldrich Chemical Company, Milwaukee WI. BSA - bovine serum albumin from Sigma Chemical Company, St. Louis MO

ELISA - enzyme linked immunosorbent assay as described in "Enzyme- Immunoassay," Edward T. Maggio, CRC Press, Inc., Boca Raton, Florida (1980) bp - base pairs ddc - dideoxycytidine g - grams mmol - millimolar

DMF - dimethyl formamide

THF - tetrahydrofuran

LSIMS - fast ion bombardment mass spectroscopy NMR - nuclear magnetic resonance spectroscopy

TMSCI - tetramethylsilylchloride

EDAC - 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride.

MES - 2-(N-morpholino)ethane sulfonic acid.

SPDP - N-succinimidyl 3-(2-pyridylthio)-propionate. -45-

Sulfo-SMCC - 4-(N-maleimidomethyl)cyclohexane-1-carboxylate. TCEP - tris-carboxyethyl phosphine.

PREPARATION OF REAGENTS C-28 thioxene:

To a solution of 4-bromoaniline (30g, 174mmol) in dry DMF (200mL) was added 1-bromotetradecane (89.3mL, 366mmol) and N,N-diisopropylethylamine (62.2mL, 357mmol). The reaction solution was heated at 90°C for 16 hr under argon before being cooled to room temperature. To this reaction solution was again added 1-bromotetradecane (45mL, 184mmol) and N,N-diisopropylethylamine (31 mL, 178mmol) and the reaction mixture was heated at 90°C for another 15 hr. After cooling, the reaction solution was concentrated in vacuo and the residue was diluted with CH₂CI₂ (400mL). The CH₂CI₂ solution was washed with 1N aqueous NaOH (2x), H₂O, and brine, was dried over Na₂SO₄ and was concentrated jn vacuo to yield a dark brown oil (about 110g). Preparative column chromatography on silica gel by a Waters 500 Prep LC system eluting with hexane afforded a yellow oil that contained mainly the product (4-bromo-N,N-di-(C-ι₄H₂₉)-aniline) along with a minor component 1-bromotetradecane. The latter compound was removed from the mixture by vacuum distillation (bp 105-110°C, 0.6mm) to leave 50.2g (51%) of the product as a brown oil. To a mixture of magnesium turnings (9.60g, 395mmol) in dry THF (30mL) under argon was added dropwise a solution of the above substituted aniline product (44.7g, 79mmol) in THF (250mL). A few crystals of iodine were added to initiate the formation of the Grignard reagent. When the reaction mixture became warm and began to reflux, the addition rate was regulated to maintain a gentle reflux. After addition was complete, the mixture was heated at reflux for an additional hour. 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-

mixture of ice water (800mL) and ethyl acetate (250mL). The organic phase was separated and the aqueous phase was extracted with ethyl acetate (3x). The combined organic phases were washed with H₂O (2x), brine and was dried over MgSO₄. Evaporation of the solvent gave 48.8g of the crude product as a dark green oily liquid. Flash column chromatography of this liquid (gradient elution with hexane, 1.5:98.5, 3:97, 5:95 ethyl acetate: hexane) afforded 24.7g (50%) of the benzoin product (LSIMS (C₄₂H₆₉NO₂): [M-H]⁺ 618.6, ¹H NMR (250 MHz, CDCI₃) was consistent with the expected benzoin product. To a solution of the benzoin product from above (24.7g, 40mmol) in dry toluene (500mL) was added sequentially 2- mercaptoethanol (25g, 320mmol) and TMSCI (100mL, 788mmol). The 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 CH₂CI₂ (3x). The combined organic layers were washed with saturated aqueous NaHCO₃ (2x) and brine, was dried over Na₂SO and was concentrated vacuo to give a brown oily liquid. Preparative column chromatography on silica gel by using a Waters 500 Prep LC system (gradient elution with hexane, 1 :99, 2:98 ethyl acetate: hexane) provided 15.5g (60%) of the C-28 thioxene as an orange-yellow oil (LSIMS (C₄₄H₇₁NOS): [M-H]⁺ 661.6, ¹H NMR (250 MHz, CDCI₃) was consistent with the expected C-28 thioxene product 2-(4-(N,N-di-(C₁ H₂₉)-anilino)-3-phenyl thioxene.

Silicon tetra-t-butyl phthalocyanine:

Sodium metal, freshly cut (5.0g, 208mmol), was added to 300mL of anhydrous methanol in a two-liter, 3-necked flask equipped with a magnetic stirrer, reflux condenser, a drying tube and a gas bubbler. After the sodium was completely dissolved, 4-t-butyl-1 ,2-dicyanobenzene (38.64g, 210mmol, from TCI Chemicals, Portland OR) was added using a funnel. The mixture became clear and the temperature increased to about 50°C. At this point a continuous stream of -47-

anhydrous ammonia gas was introduced through the glass bubbler into the reaction mixture for 1 hr. The reaction mixture was then heated under reflux for 4 hr. while the stream of ammonia gas continued. During the course of the reaction, as solid started to precipitate. The resulting suspension was evaporated to dryness (house vacuum) and the residue was suspended in water (400mL) and filtered. The solid was dried (60°C, house vacuum, P₂O₅). The yield of the product (1 ,3- diiminoisoindoline, 42.2g) was almost quantitative. This material was used for the next step without further purification. To a one-liter, three-necked flask equipped with a condenser and a drying tube was added the above product (18g, 89mmol) and quinoline (200mL, Aldrich Chemical Company, St. Louis MO). Silicon tetrachloride (11mL, 95mmol, Aldrich Chemical Company) was added with a syringe to the stirred solution over a period of 10 minutes. After the addition was completed, the reaction mixture was heated to 180-185°C in an oil bath for 1 hr. The reaction was allowed to cool to room temperature and concentrated HCI was carefully added to acidify the reaction mixture (pH 5-6). The dark brown reaction mixture was cooled and filtered. The solid was washed with 100mL of water and dried (house vacuum, 60°C, P₂O₅). 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. The solid was washed with 50mL of water and dried under vacuum (house vacuum, 60°C, P₂O₅) to give 12g of product silicon tetra- t-butyl phthalocyanine as a dark blue solid. 3-picoline (12g, from Aldrich Chemical Company), tri-n-butyl amine (anhydrous, 40mL) and tri-n-hexyl chlorosilane (11.5g) were added to 12g of the above product in a one-liter, three-necked flask, equipped with a magnetic stirrer and a reflux condenser. The mixture was heated under reflux for 1.5 hr. and then cooled to room temperature. The picoline was distilled off under -48-

high vacuum (oil pump at about 1 mm Hg) to dryness. The residue was dissolved in CH₂CI₂ and purified using a silica gel column (hexane) to give 10g of pure product di- (tri-n-hexylsilyl)-silicon tetra-t-butyl phthalocyanine as a dark blue solid. (LSIMS: [M- H]⁺ 1364.2, absorption spectra: methanol: 674nm (ε 180,000): toluene 678nm, ¹H NMR (250 MHz, CDCI₃): δ: -2.4(m,12H), -1.3(m, 12H), 0.2-0.9 (m, 54H), 1.8(s, 36H), 8.3(d, 4H) and 9.6 (m, 8H) was consistent with the above expected product.

Hydroxypropylaminodextran: (1 NH₂/ 7 glucose) was prepared by dissolving Dextran T-500 (Pharmacia, Uppsala, Sweden) (50g) in 150 mL of H₂O in a 3-neck round-bottom flask equipped with mechanical stirrer and dropping funnel. To the above solution was added 18.8g of Zn (BF₄)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 NH₂/ 7 glucose), prepared above, was dissolved in 50mM MOPS, pH 7.2, at 12.5 mg/mL. The solution was stirred for 8 hr at room temperature, stored under refrigeration and centrifuged for 45 min at 15,000 rpm in a Sorvall RC-5B centrifuge immediately before use to remove a trace of solid material. To 10mL of this solution was added 23.1 mg of Sulfo-SMCC in 1mL of -49-

water. This mixture was incubated for 1 hr at room temperature and used without further purification.

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. Following the 20-minute incubation, 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., Laguna 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-

Oligonucleotide Bound Chemiluminescer Particles:

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 H₂O). The sulfo-SMCC was added to a solution of the aminodextran (in 40 mL, 0.05 M Na₂HPO , 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.

Thiolated oligonucleotide (oligonucleotide bearing a 5'-bis(6- hydroxyethyldisulfide) group) (Oligos Etc.) was dissolved in water at a concentration of 0.49 mM. To 116 μL of this solution was added 8.3 μL of 3.5 M sodium acetate, pH 5.3 and 8.9 μL of tris(carboxyethyl)phosphine (20 mM). After 30 minutes incubation at room temperature, 548 μL 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 μL of 5mM sodium phosphate, 2 mM EDTA, pH 6. -51-

An aliquot of the maleimidated beads prepared above containing 22 mg beads was centrifuged for 30 min. at about 37,000 g, and the pellet was resuspended in 96 μL of 0.26 M NaCI, 0.05% Tween-20, 95 mM sodium phosphate, and 0.95 mM EDTA, pH7. The thiolated oligonucleotide was added and the mixture was maintained at 37°C for 64 hours under argon. A 10 μL aliquot of sodium thioglycolate was added and incubation was continued for 2 hours at 37°C. Water was added to a total volume of 1 mL, and the beads were recovered by centrifugation, then resuspended in 5 mL of 0.1 M NaCI, 0.17 M glycine, 10 mg/mL BSA, 1 mM EDTA, 0.1 % Tween-20, and 0.5 mg/mL Calf thymus DNA (Sigma Molecular Biology grade), pH 9.2. After three hours, the beads were recovered and washed three times by centrifugation, twice in buffer A and once in standard PCR buffer. The product was stored refrigerated in PCR buffer. Buffer A contained 0.1 M Tris base (J.T. Baker Chemical Co.), 0.3 M NaCI (Mallinckrodt), 25 mM EDTA Na₂ H₂O (Sigma Chemical Co.), 0.1 % BSA (Sigma Chemical Co.), 0.1 % dextran (Pharmacia), HBR-1 (Scantibodies), 0.05% Kathon and 0.01 % gentamicin sulfate (GIBCO) prepared by dissolving and adjusting pH to 8.20 with concentrated HCI and made up to 10 L with distilled water.

The sequence of the oligonucleotide bound to the chemiluminescer particles was 5'-(AGTA)₆. The above procedure may be modified in a manner similar to that described by Ullman, et al., Proc. Natl. Acad. Sci. USA (1994) 9J.:5426-5427 at column 1 of page 5427. Sensitizer Particles:

Four mL of 20% suspension (400 mg) of washed 175 nm carboxylate modified latex was diluted with 3 mL of ethoxyethanol in a 25 mL round bottom (R.B.) flask with a stir bar. The R.B. flask was then placed in an oil bath at 105°C and stirred for 10 minutes. Then, 40 mg of silicon tetra-t-butyl phthalocyanine prepared as described above was added; the beads were stirred for 5 minutes more. At this point 1.0 mL of 0.1 N NaOH was added slowly over 5 minutes. During all the -52-

additions, the oil bath temperature was maintained at 105°C. The oil bath temperature was slowly allowed to drop to room temperature over 2 hours. After cooling, 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 dA₂ .

Example 1 The approach utilized in this example is depicted in Fig. 4. In this example a complex of second oligonucleotide probe PPP composed of a sequence PPcι_1 at the 5'-end and a sequence PP_SN2 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_CL1 of the oligonucleotide of the chemiluminescer particle reagent P_CL, which corresponds to a first oligonucleotide probe, and 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. In addition to TS3, 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-

compound decreases. Thus, the more target polynucleotide present, the greater the displacement and the larger the decrease in signal.

In another embodiment a different target polynucleotide TS' was employed in place of TS. 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. In this embodiment 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. In addition to TS3, 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_Cι_-PPP. As a result of the displacement, the signal generated by the chemiluminescent compound decreases. Thus, 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)_fiCATGAATGGGATAGAGTGCATCCAGTG-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:

AGTA3'-(ATGA)₆ (SEQ ID NO:3) (negative control) PL-2: 3'-(ATGA)_R-CTTACCCTATCTCACGTAGGTCAC (SEQ ID NO:4) PL-3: 3'-CTTACCCTATCTCACGTAGGTCAC-(A)_?4 (SEQ ID NO:5) A24: 3'-(A)₂₄ (SEQ ID NO:6) (negative control) -54-

The underlined sequence (TS3 and TS'3, respectively) was complementary to the underlined sequence P'P'3 of the second oligonucleotide probe. The two systems, with and without the additional CAT in the second oligonucleotide probe, were tested to demonstrate displacement and to demonstrate that displacement was not affected by a gap of a few nucleotides (i.e., 3 nucleotides in this example). 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_L1 ) immobilized through the 3'-end and the sensitizer particles had dA₂₄ (SEQ ID NO:8) (P_SN1 ) immobilized thereon through the 3'-end. The particles were mixed with OL-1 or OL-4 to a final concentration of 1 μg of each of the particles and 5 pM linker, in a total volume of 39 μl in NL buffer with 15% DMSO (10mM Tris-HCL, 70 mM KCI, 12mM MgCI₂, 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.

-55-

Table 2

Targets Target Linker concentration

None OL-1 OL-4

None 366 81926 80618

PL-2 3.75pM 416 83290 84016

PL-3 3.75pM 398 82800 82084

PL-2 15pM 400 78584 77848

PL-3 15pM 448 67464 71704

PL-2 37.5pM 414 62930 61562

PL-3 37.5pM 398 44080 47496

PL-2 1500pM 406 452 474

The above results demonstrate that both targets, PL-2 and PL-3 equally displaced the second oligonucleotides, OL-1 and OL-4, respectively, for the preformed complex. In addition, the displacement, as indicated by the decrease in signal, was dependent on target concentration.

Example 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 μg of each of the particles and 5.9 pM for the linker, in a total volume of 45 μl in NL buffer. Mineral oil (20 μl) 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 μl of the target was added. The displacement of the preformed particle/linker -56-

complex was measured by measuring the signal. Reaction tubes were irradiated for 0.1 sec. and read for 1 sec. The results are summarized in the following Table 3.

Table 3

Target Linker Minutes after addition of target to preformed compli ex

M 50DM) (4.9pM) 0 2 5 10 20 30 150

H₂O OL-1 83513 84733 86847 87454 88075 88280 88117

(AGTA)₆ OL-1 82555 84686 86276 87015 87450 86773 86909

PL-2 OL-1 82081 82560 83318 82379 76929 62916 34728

H₂O OL-4 85736 88749 89608 91257 90528 90990 91573

A₂₄ OL-4 79249 80516 81484 81868 81459 80564 77052

PL-3 OL-4 84764 86127 85066 81761 74994 60187 32436

The above results demonstrate that both targets displaced the second oligonucleotide from the preformed complex with the oligonucleotide particles and the displacement is equally efficient. In the case of OL-4, for example, the inhibition was clearly dependent on strand displacement as sequences complementary to the second oligonucleotides binding to the oligonucleotide particles, namely, A₂₄ in the for OL-4, did not displace the preformed second oligonucleotide particle complexes. The results also demonstrate that in the case of OL-1 , for example, the inhibition was similarly dependent on strand displacement since a sequence complementary to the second oligonucleotide binding to the oligonucleotide particles, namely, (ATGA)β, did not displace the preformed second oligonucleotide particle complexes.

The above discussion includes certain theories as to mechanisms involved in the present invention. These theories should not be construed to limit the present invention in any way, since it has been demonstrated that the present invention achieves the results described.

The above description and examples fully disclose the invention including preferred embodiments thereof. Modifications of the methods described that are obvious to those of ordinary skill in the art such as molecular biology and related sciences are intended to be within the scope of the following claims.

Claims

-57-WHAT IS CLAIMED IS:

1. A method for detecting a single stranded target polynucleotide, which comprises:

(a) providing in combination (i) a medium suspected of containing said single stranded target polynucleotide, (ii) a first oligonucleotide probe and (iii) a second oligonucleotide probe wherein (I) said first probe has a sequence P1 that is capable of hybridizing with a sequence PP1 of said second probe, (II) said 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 said second probe, (III) said first probe is incapable of hybridizing to said PP2 or said single stranded target polynucleotide, (IV) S1 and S2 or PP1 and PP2 are noncontiguous and (V) one or both of said first probe and said second probe comprise members of a signal producing system such that the binding of said single stranded target polynucleotide, if present, to said second probe alters a sjgnal generated by said signal producing system;

(b) subjecting said combination to conditions under which said single strand, if present, hybridizes to said second probe and displaces said first probe; and

(c) detecting said signal.

2. A method for amplifying and detecting a target polynucleotide, which comprises:

(a) providing in combination (i) a medium suspected of containing said target polynucleotide, (ii) all reagents required for conducting an amplification of said target polynucleotide to produce an amplification product, and (iii) first and second oligonucleotide probes wherein (I) said first probe has a sequence P1 that is capable of hybridizing with a sequence PP1 of said second probe, (II) said 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 said second probe, (III) said first probe is incapable of hybridizing to said PP2 or said -58-

single strand, (IV) S1 and S2 or PP1 and PP2 are noncontiguous and (V) one or both of said first probe and said second probe comprise members of a signal producing system such that the binding of said single stranded amplification product, if present, to said second probe alters a signal generated by said signal producing system;

(b) subjecting said combination to conditions for amplifying said target polynucleotide;

(c) subjecting said combination to conditions under which said single stranded amplification product, if present, hybridizes to said second probe and displaces said first probe from said second probe; and

(d) detecting said signal.

3. The method of Claim 2 wherein said signal is detected and related to the concentration of said amplification product.

4. The method of Claim 2 wherein said amplification is carried out at an isothermal temperature and a change in said signal is monitored during said amplification to determine the concentration of said amplification product.

5. The method of Claim 2 wherein one of said first and second probes is labeled with a sensitizer or a quencher.

6. The method of Claim 2 wherein one of said first and second probes is labeled with a chemiluminescent compound or a fluorescent compound.

7. The method of Claim 2 wherein said amplification is selected from the group consisting of NASBA, 3SR, SDA and amplifications utilizing Q╬▓-replicase.

8. The method of Claim 2 wherein said polynucleotide is DNA. -59-

9. The method of Claim 2 wherein said polynucleotide is RNA.

10. The method of Claim 2 wherein PP1 and PP2 are separated by a sequence of 1 to 30 nucleotides.

11. The method of Claim 2 wherein S1 and S2 are separated by a sequence of 1 to 100 nucleotides.

12. The method of Claim 2 wherein S1 and S2 are contiguous.

13. A method for amplifying and detecting a plurality of different polynucleotides, which comprises:

(a) providing in combination (i) a medium containing a plurality of different polynucleotides, (ii) all reagents required for conducting an amplification of said 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 for each of said different amplification products wherein for each of said sets (I) said first probe has a sequence P1 that is capable of hybridizing with a sequence PP1 of said second probe, (II) said 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 said second probe, (III) said first probe is incapable of hybridizing to said PP2 or said single strand, (IV) S1 and S2 or PP1 and PP2 are noncontiguous and (V) one or both of said first probe and said second probe comprise members of a signal producing system unique to each of said sets such that the binding of said single stranded amplification product to said second probe alters a signal generated by said signal producing system, wherein the combination of the sequence pair S1 and PP1 and the sequence pair S2 and PP2 is unique in each of said sets and the signals generated by each of said signal producing systems are differentially detectable; -60-

(b) subjecting said combination to conditions for amplifying said polynucleotides;

(c) subjecting said combination to conditions under which each of said single stranded amplification products hybridizes to its respective second probe and displaces its respective first probe; and

(d) differentially detecting each of said detectable signals.

14. The method of Claim 13 wherein said signals are related to the concentration of said amplified products in said medium.

15. The method of Claim 14 wherein the concentration of all but one of said plurality of polynucleotides is known and the concentration of the remaining of said polynucleotides is determined by comparing the signal associated with its amplified product with the signals associated with the other of said amplified products.

16. The method of Claim 13 wherein said signal producing systems comprise labels selected from the group consisting of a luminescent energy donor and acceptor pair, a singlet oxygen generator and chemiluminescent reactant pair, and an enzyme pair wherein a product of the first enzyme serves as a substrate for the second enzyme.

17. The method of Claim 13 wherein said amplification is carried out at an isothermal temperature and changes in said signals are monitored during said amplification to determine the concentration of said amplification product.

18. The method of Claim 13 wherein for each of said sets one of said first and second probes is labeled with a sensitizer. -61-

19. The method of Claim 13 wherein for each of said sets one of said first and second probes is labeled with a chemiluminescent compound.

20. The method of Claim 13 wherein said amplification is selected from the group consisting of NASBA, 3SR, SDA and amplifications utilizing Q╬▓-replicase.

21. The method of Claim 13 wherein said polynucleotide is DNA.

22. The method of Claim 13 wherein said polynucleotide is RNA.

23. The method of Claim 13 wherein for each of said sets PP1 and PP2 are separated by a sequence of 1 to 30 nucleotides.

24. The method of Claim 13 wherein for each of said sets S1 and S2 are separated by a sequence of 1 to 100 nucleotides.

25. The method of Claim 13 wherein for each of said sets S1 and S2 are contiguous.

26. A method for amplifying and detecting a plurality of different RNA's, which comprises:

(a) providing in combination (i) a medium containing a plurality of different RNA's, (ii) all reagents required for conducting an amplification of said 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 for each of said different amplification products, wherein for each of said sets (I) said first probe has a sequence P1 that is capable of hybridizing with a sequence PP1 of said second probe, (II) said 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 said second probe, (III) said first probe is incapable of hybridizing to said PP2 -62-

or said RNA strand, (IV) S1 and S2 or PP1 and PP2 are noncontiguous and (V) one of said probes is labeled with a sensitizer and the other of said probes is labeled with a chemiluminescer such that the binding of said amplification product to said second probe alters the chemiluminescence generated by said chemiluminescer wherein the combination of the sequence pair S1 and PP1 and the sequence pair S2 and PP2 is unique in each of said sets and respective labels for each of said sets are differentially detectable;

(b) subjecting said combination to isothermal conditions for amplifying said RNA's; (c) subjecting said combination to conditions under which each of said amplification products hybridizes to its respective second probe and displaces its respective first probe; and

(d) differentially detecting each of said detectable signals.

27. The method of Claim 26 wherein said signals are related to the concentrations of said amplified products in said medium.

28. The method of Claim 26 wherein said signals are detected and related to the concentrations of said amplification products.

29. The method of Claim 26 wherein said signals are monitored during said amplification to determine the concentrations of said amplification products.

30. The method of Claim 26 wherein said sensitizer is a photosensitizer.

31. The method of Claim 26 wherein said chemiluminescer is a particle containing a singular oxygen reactive olefin. -63-

32. The method of Claim 26 wherein PP1 and PP2 are separated by a sequence of 1 to 30 nucleotides.

33. The method of Claim 26 wherein S1 and S2 are separated by a sequence of 1 to 100 nucleotides.

34. The method of Claim 26 wherein S1 and S2 are contiguous.

35. A kit for use in amplification and detection of a target polynucleotide comprising in packaged combination:

(a) reagents for conducting an amplification of said target polynucleotide, and

(b) first and second oligonucleotide probes wherein (I) said first probe has a sequence P1 that is capable of hybridizing with a sequence PP1 of said second probe, (II) said 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 said second probe, (III) said first probe is incapable of hybridizing to said PP2 or said single strand, (IV) S1 and S2 or PP1 and PP2 are noncontiguous and (V) one or both of said first probe and said second probe comprise members of a signal producing system such that the binding of said single stranded amplification product, if present, to said second probe alters a signal generated by said system.

36. The kit of Claim 35 wherein said signal producing system comprises a label on one of said first or said second probes wherein said label is a particle having a chemiluminescent compound associated therewith.

37. The kit of Claim 35 wherein said signal producing system comprises a label on one of said first or said second probes wherein said label is a sensitizer. -64-

38. The kit of Claim 36 wherein said reagents for conducting an amplification comprise a promoter and an enzyme.

39. The kit of Claim 36 wherein said amplification is selected from the group consisting of NASBA, 3SR, SDA and amplifications utilizing Q╬▓-replicase.

40. The kit of Claim 36 wherein said target polynucleotide is DNA.

41. The kit of Claim 36 wherein said target polynucleotide is RNA.

42. The kit of Claim 36 wherein PP1 and PP2 are separated by a sequence of 1 to 30 nucleotides.

43. The kit of Claim 36 wherein S1 and S2 are separated by a sequence of 1 to 100 nucleotides.

44. The kit of Claim 36 wherein S1 and S2 are contiguous.

45. A kit for use in an amplification and quantitative of a specific RNA, said kit comprising in packaged combination: (a) one or more reference RNA's,

(b) a promoter,

(c) an enzyme and

(d) a plurality of sets of first and second oligonucleotide probes, one set for each different RNA , wherein for each of said sets (i) said first probe has a sequence P1 that is capable of hybridizing with a sequence PP1 of said second probe, (ii) said 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 said second probe, (iii) said first probe is incapable of hybridizing to said PP2 or said -65-

single strand, (iv) S1 and S2 or PP1 and PP2 are noncontiguous and (v) one of said probes is labeled with a sensitizer and the other of said probes is labeled with a chemiluminescer wherein the combination of the sequence pair S1 and PP1 and the sequence pair S2 and PP2 is unique in each of said sets and the respective labels for each of said sets are differentially detectable.

46. The kit of Claim 45 wherein said sensitizer is a photosensitizer.

47. The kit of Claim 45 where said chemiluminescer is a singlet oxygen reactive olefin.

48. The kit of Claim 45 wherein PP1 and PP2 are separated by a sequence of 1 to 30 nucleotides.

49. The kit of Claim 45 wherein S1 and S2 are separated by a sequence of

1 to 100 nucleotides.

50. The kit of Claim 45 wherein S1 and S2 are contiguous.