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WO2001090399A2 - Detection de mutations et de polymorphismes dans des acides nucleiques - Google Patents

Detection de mutations et de polymorphismes dans des acides nucleiques Download PDF

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Publication number
WO2001090399A2
WO2001090399A2 PCT/US2001/016089 US0116089W WO0190399A2 WO 2001090399 A2 WO2001090399 A2 WO 2001090399A2 US 0116089 W US0116089 W US 0116089W WO 0190399 A2 WO0190399 A2 WO 0190399A2
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Prior art keywords
probe
sequence
polynucleotide
label
signal producing
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PCT/US2001/016089
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English (en)
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Rajesh D. Patel
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Dade Behring Inc.
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Publication of WO2001090399A2 publication Critical patent/WO2001090399A2/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

Definitions

  • This application relates to the detection of mutations and polymorphisms in nucleic acids.
  • the mutations may be single or multiple nucleotide polymorphisms, or insertions or deletions of one or more nucleotides in a polynucleotide sequence.
  • the detection involves the use of competitive probes and multiple signals and is accomplished with or without nucleic acid amplification.
  • nucleotide position is viewed as polymorphic when it exists in at least two variants and the allele frequency of the most common variant is less than 99% (4).
  • Extensive sets of polymorphic sequences are now being identified in the course of genome research, in studies of human genome diversity, and in the process of establishing mutation databases that may ultimately include all common variants of human genes (5).
  • Applications of polymorphism analysis now extends from investigations of small numbers of sequence variants known to be associated with a specific disease to investigation of markers across the genome, perhaps with markers corresponding to several variants of each of the roughly 100,000 human genes.
  • a reliable technique of analyzing mutations in genes of medical interest for disease diagnosis and the prediction of pharmaceutical efficacy requires the ability to discriminate a difference in a single nucleotide.
  • methods of analyzing DNA sequences have been restricted largely to research and specialized clinical laboratories due to their technological complexity and cost. These methods include reverse oligonucleotide hybridization, competitive or allele- specific PCR, and ligase chain reaction. These techniques are useful for detecting the mutations and hereditary diseases when 50% or 100% of the analyzed DNA contains the mutation.
  • a more discriminative procedure is needed to detect somatic mutations, such as human malignancies, where very often only a small fraction of DNA contains the mutation.
  • each of the techniques established so far has shortcomings.
  • Reverse oligonucleotide hybridization involves multiple, labor-intensive steps including DNA template amplification, immobilization of the DNA template to a solid support, hybridization, washing and detection (6, 7), making it difficult to automate.
  • the C-PCR (8) and LCR (9-11 ) techniques are based on competitive priming and amplification, however, they are very prone to mismatched priming and exponential amplification of the mismatched hybrids (10, 12, 13), resulting in high background or false positive results. In the case of LCR, the inherent blunt-end ligation activity of ligases is another source of error (13, 14).
  • High-throughput detection methods for single nucleotide variations currently in use include homogeneous methods such as template-directed dye-terminator incorporation (TDI) assay (23), the 5'-nuclease allele-specific hybridization TaqMan assay (24), and the recently described allele-specific molecular beacon assay (25).
  • the TDI assay combines PCR and specific primer extension with fluorescence resonance energy transfer (FRET) detection and requires almost no optimization, but requires three separate steps that include adding reagents twice after the initial reaction set up (26).
  • the TaqMan assay is the simplest of all diagnostic assays in which one can determine the mutational status of a DNA sample in one step during PCR (24).
  • the TaqMan assay has the stringent requirement that a perfectly complimentary internal probe hybridizes and is cleaved at the polymorphic site during strand extension phase in PCR, whereas the corresponding probe with a 1-base mismatch does not. This requires some optimization and may be difficult to achieve in AT-rich domains. Allele discrimination by the use of molecular beacons of four different colors has been shown to work when synthetic oligonucleotides are used as targets, but assay conditions for amplified and genomic DNA targets are still being worked out (25). Other methods include the dye labeled oligonucleotide ligation (DOL) which combines PCR and an oligonucleotide ligation reaction in a two-stage thermal cycling sequence with FRET. The FRET detection occurs when a donor and acceptor dye are in close proximity.
  • DOL dye labeled oligonucleotide ligation
  • One aspect of the present invention is a method for detecting the presence of a polynucleotide analyte.
  • the method comprises combining in a reaction medium the polynucleotide analyte and three oligonucleotide probes.
  • the first probe comprises a sequence complementary to a first region of the polynucleotide analyte.
  • the second probe comprises a sequence complementary to a second region of the polynucleotide analyte.
  • the third probe comprises a sequence complementary to the second region except for the difference of at least one nucleotide, or the insertion or deletion of at least one nucleotide.
  • the reaction medium is subjected to conditions for forming substantially non-dissociative termolecular complexes of at least one of (i) the polynucleotide analyte, the first probe and the second probe or (ii) the polynucleotide analyte, the first probe and the third probe.
  • the presence of at least one of the termolecular complexes is determined.
  • the first, second and third probes are labeled with first, second and third labels, respectively.
  • the first label comprises a first member of first and second signal producing systems
  • the second label comprises a second member of the first signal producing system
  • the third label comprises a second member of the second signal producing system.
  • the labels are non-covalently bound to the probes, and more preferably, through nucleic acid hybridization.
  • the first, second and third oligonucleotide probes include second sequences which do not hybridize to the polynucleotide analyte or each other.
  • the first label further comprises a sequence which hybridizes to the second sequence of the first probe
  • the second label comprises a sequence which hybridizes to the second sequence of the second probe
  • the third label comprises a sequence which hybridizes to the second sequence of the third probe.
  • the labels include members of signal producing systems such as 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.
  • the first member of the signal producing systems is a sensitizer when the second members of the signal producing systems are chemiluminescent compounds.
  • the first member of the signal producing systems may be a chemiluminescent compound when the second members of the signal producing systems are sensitizers.
  • the chemiluminescent compounds are preferably enol ethers, enamines, 9- alkylidene-N-alkylacridans, arylvinylethers, dioxenes, arylimidazoles, 9-alkylidene- xanthanes and lucigenin.
  • the sensitizers are preferably photosensitizers, and more preferably photosensitizers such as methylene blue, rose bengal, porphyrins and phthalocyanines or naphtholcyanines.
  • a further aspect of the invention provides for a method for detecting the presence of one or both of a first and a second polynucleotide analytes which differ from each other by one or more nucleotides or by the insertion or deletion of at least one nucleotide.
  • the method involves combining in a reaction medium a sample suspected of containing one or both polynucleotide analytes, and three oligonucleotide probes.
  • the first probe comprises a sequence which hybridizes to a sequence common to both the first and second polynucleotide analytes.
  • the second probe comprises a sequence which hybridizes to a second region of first polynucleotide analyte.
  • the third probe comprises the same sequence as the second probe except for a difference of at least one nucleotide, or the insertion or deletion of at least one nucleotide.
  • the difference, the insertion or the deletion represents the difference, insertion or deletion between the first and second polynucleotide analytes.
  • the reaction medium is subjected to conditions forming at least one of (i) a first termolecular complex comprising the first polynucleotide analyte, the first probe and the second probe and, (ii) a second termolecular complex comprising the second polynucleotide analyte, the first probe and the third probe. The presence of at least one of the first and second termolecular complexes is then detected.
  • reaction medium includes reagents sufficient for amplifying the polynucleotide analyte and the medium is subjected to conditions for amplifying the polynucleotide analyte.
  • One of the preferred embodiments of the present invention includes a method for detecting the presence of a single nucleotide polymorphism in a fragment of genomic DNA: The method includes combining in a reaction medium a fragment of genomic DNA suspected of containing a single nucleotide polymorphism and three oligonucleotide probes.
  • the first probe comprises a first label and a sequence which hybridizes to a first region of the polynucleotide analyte.
  • the second probe comprises a second label and sequence which hybridizes to a second region of the polynucleotide analyte.
  • the third probe comprises a third label and the same sequence as the sequence of the second probe except for a difference of one nucleotide, wherein the difference is complementary to a single nucleotide polymorphism in the second sequence.
  • the reaction medium is subjected to conditions for annealing the first probe, the second probe and the third probe to the polynucleotide analyte.
  • the interaction of the first label and the second label produces a first signal and the interaction of the first label and the third label produces a second signal. Each of the signals is detected.
  • Another embodiment of the present invention provides a method for detecting the presence of two or more polynucleotide analytes wherein the analytes are alleles which differ from each other by one or more nucleotides or the insertion or deletion of one or more nucleotides.
  • the method includes combining in a reaction medium the polynucleotide analytes and at least three oligonucleotide probes.
  • the first probe comprises a sequence complementary to a first region common to each of the the polynucleotide analytes.
  • the second probe comprises a sequence complementary to a second region of one of the polynucleotide analyte.
  • One or more third probes comprise a sequence complementary to the second region except for a difference of at least one nucleotide, or the insertion or deletion of at least one nucleotide.
  • the difference, insertion or deletion corresponds to the one or more alleles.
  • the combination is subjected to conditions for forming substantially non-dissociative termolecular complexes of at least one of (i) the polynucleotide analyte, the first probe and the second probe or (ii) the polynucleotide analyte, the first probe and the one or more third probes.
  • the presence of at least one of the termolecular complexes is determined.
  • a further aspect of the present invention includes a kit for use in a determination of a target polynucleotide in a sample.
  • the kit includes a first oligonucleotide probe comprises a sequence which hybridizes to a first region of the target polynucleotide; a second oligonucleotide probe which hybridizes to a second region of the target polynucleotide; and a third oligonucleotide probe comprising the same sequence as the sequence of the second probe which hybridizes to the second region of the target polynucleotide except for the difference of one or more nucleotides or the insertion or deletion of one or more nucleotides, the difference, insertion or deletion being complimentary to an expected mutation of the target polynucleotide.
  • the kit also includes a first label comprising a first member of first and second signal producing systems, a second label comprising a second member of the first signal producing system, and a third label comprising a second member of the second signal producing system.
  • the first second and third labels can non-covalently bind to the first second and third oligonucleotide probes, respectively.
  • the kit may also include reagents for conducting an amplification of the polynucleotide analyte.
  • 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.
  • Figs. 3, 4 and 5 are graphs showing results of assays in accordance with the present invention for the detection of mismatches and mutations in nucleic acid sequences.
  • the present invention can be used to detect the presence of a mutation in a polynucleotide sequence, or to detect differences between polynucleotide sequences, where the mutation or difference can be a single nucleotide polymorphism, a polymorphism of more than one nucleotide, or the insertion or deletion of one or more nucleotides.
  • the method uses at least two probes having nucleotide sequences which are capable of hybridizing to a target polynucleotide distinguished from each other by the one or more nucleotides, representing the difference between a wild type sequence (reference) and a sequence containing the difference or mutation (target or mutant sequence).
  • the probes can each fully anneal to either a target or reference polynucleotide sequence that may be present in a sample.
  • a sample contains a target or reference polynucleotide sequence exactly complementary to a sequence of one of the probes, the probe will hybridize to the target or reference sequence and strongly compete with and inhibit the hybridization of the mismatched probe.
  • both probes will bind to the sequences to which they are fully complementary and inhibit the binding of the mismatched probe.
  • the hybridization of either or both probes can be distinguished in a homogenous format using a different signal producing system for each probe. The method may be carried out with or without amplification of the target and reference sequences.
  • the polynucleotide analytes 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 microorganisms (e.g., bacteria, yeasts, viruses, viroids), molds, fungi, plants, animals, humans, and the like.
  • DNA dsDNA and ssDNA
  • 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 microorganisms (e.g., bacteria, yeasts, viruses, viroids
  • 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.
  • the polynucleotide analyte can be any nucleotide sequence including a synthetic polynucleotide but is most often a wild type nucleic acid sequence or a mutant nucleic acid sequence.
  • a wild type sequence being most often found in the genome of an organism.
  • a mutant sequence having differences of one or more nucleotides or insertions or deletions of one or more nucleotides, compared to the wild type sequence. For any particular sequence of any particular organism, more than one wild type or mutant sequence may exist.
  • the polynucleotide analyte may be cleaved to obtain a fragment that contains a target polynucleotide sequence, for example, by shearing or by treatment with a restriction endonuclease or other site specific chemical cleavage method.
  • the polynucleotide analyte, or a cleaved fragment obtained from the polynucleotide analyte will usually be at least partially denatured or single stranded or treated to render it denatured or single stranded.
  • treatments include, for instance, heat or alkali treatment.
  • double stranded DNA can be heated at 90-100° C. for a period of about 1 second to about 10 minutes to produce denatured material.
  • Mutation - a difference of one or more nucleotides, or the insertion of deletion of one or more nucleotides between two polynucleotide sequences.
  • Mutation includes a single nucleotide polymorphism ("SNP") which is the difference of a single nucleotide between two polynucleotide sequences.
  • SNP single nucleotide polymorphism
  • 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 in 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 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 involves amplification of a single stranded polynucleotide using a single oligonucleotide primer.
  • the single stranded polynucleotide that is to be amplified contains two non-contiguous sequences that are complementary to one another and, thus, are capable of hybridizing together to form a stem-loop structure.
  • This single stranded polynucleotide already may be part of a polynucleotide analyte or may be created as the result of the presence of a polynucleotide analyte.
  • LCR ligase chain reaction
  • NASBA nucleic acid sequence based amplification
  • the reagents for conducting NASBA include a first DNA primer with a 5' tail comprising a promoter, a second DNA primer, reverse transcriptase, RNAse-H, T7
  • RNA polymerase 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.
  • 3SR Sequence Replication
  • RNAse-H activity is present in the reverse transcriptase.
  • Amplification by 3SR is an RNA specific target method whereby RNA is amplified in an isothermal process combining promoter directed RNA polymerase, reverse transcriptase and RNase
  • TMA Transcription Mediated Amplification
  • 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.
  • the primary difference between linear amplification and exponential amplification is that the latter is autocatalyzed, that is, the product serves to catalyze the formation of more product, whereas in the former process the starting sequence catalyzes the formation of product but is not itself replicated.
  • linear amplification the amount of product formed increases as a linear function of time as opposed to exponential amplification where the amount of product formed is an exponential function of time.
  • Target polynucleotide a sequence of nucleotides to be identified, usually existing within a portion or all of a polynucleotide analyte, the identity of which is known to an extent sufficient to allow preparation of various oligonucleotides, such as probes and primers, and other molecules necessary for conducting an amplification of the target polynucleotide.
  • 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 or amplicons.”
  • the target sequence usually lies between two defined sequences but need not.
  • the primers hybridize with the defined sequences or with at least a portion of such target polynucleotide, usually at least a ten-nucleotide segment at the 3'- end thereof and preferably at least 15, frequently 20 to 50 nucleotide segment thereof.
  • the target sequence usually contains from about 30 to 5,000 or more nucleotides, preferably 50 to 1 ,000 nucleotides.
  • the target polynucleotide is generally a fraction of a larger molecule or it may be substantially the entire molecule (polynucleotide analyte).
  • the minimum number of nucleotides in the target polynucleotide sequence is selected to assure that the presence of target polynucleotide in a sample is a specific indicator of the presence of polynucleotide analyte in a sample.
  • the sequence length is usually greater than about 1.6 log L nucleotides where L is the number of base pairs in the genome of the biologic source of the sample.
  • the maximum number of nucleotides in the target polynucleotide is normally governed by the length of the polynucleotide analyte and its tendency to be broken by shearing, or other processes during isolation and any procedures required to prepare the sample for assay and the efficiency of detection and/or amplification of the sequence.
  • Oligonucleotide - a polynucleotide, usually single stranded, usually a synthetic polynucleotide but may be a naturally occurring polynucleotide.
  • the oligonucleotide(s) are usually comprised of a sequence of at least 5 nucleotides, preferably, 10 to 100 nucleotides, more preferably, 20 to 50 nucleotides, and usually 10 to 30 nucleotides in length.
  • Various techniques can be employed for preparing an oligonucleotide utilized in the present invention.
  • Such oligonucleotide can be obtained by biological synthesis or by chemical synthesis. 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.
  • 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.
  • oligonucleotide synthesis include phosphotriester and phosphodiester methods (Narang, et al. (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, 154:287-314 (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 sequence which binds to the target polynucleotide can be DNA, RNA, peptide nucleic acids (PNA) or any sequence which is capable of specifcally binding.
  • PNA peptide nucleic acids
  • 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 herein.
  • 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.
  • nucleoside triphosphates also includes derivatives and analogs thereof, which are exemplified by those derivatives that are recognized in a similar manner to the unde vatized nucleoside triphosphates.
  • 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.
  • the catalysts are enzymes, such as DNA polymerases, for example, prokaryotic DNA polymerase (I, II, or III), T4 DNA polymerase, T7 DNA polymerase, Klenow fragment, reverse transcriptase, Vent DNA polymerase, Pfu DNA polymerase, Taq DNA polymerase, and the like, derived from any source such as cells, bacteria, such as E. coN, plants, animals, virus, thermophilic bacteria, and so forth.
  • RNA polymerases include T7 RNA polymerase, AMV polymerase, Q-beta-replicase, and so forth.
  • Hybridization and binding - in the context of nucleotide sequences these terms are used interchangeably herein.
  • the ability of two nucleotide sequences to hybridize with each other is based on the degree of complementarity of the two nucleotide sequences, which in turn is based on the fraction of matched complementary nucleotide pairs.
  • the more nucleotides in a given sequence that are complementary to another sequence the more stringent the conditions can be for hybridization and the more specific will be the binding of the two sequences.
  • Increased stringency is achieved by elevating the temperature, increasing the ratio of cosolvents, lowering the salt concentration, and the like.
  • Homologous or substantially identical polynucleotides In general, two polynucleotide sequences that are identical or can each hybridize to the same polynucleotide sequence are homologous. The two sequences are homologous or substantially identical where the sequences each have at least 90%, preferably 100%, of the same or analogous base sequence where thymine (T) and uracil (U) are considered the same. Thus, the ribonucleotides A, U, C and G are taken as analogous to the deoxynucleotides dA, dT, dC, and dG, respectively.
  • Homologous sequences can comprise DNA, RNA or modified polynucleotides and may be homoduplexes, e.g., RNA:RNA and DNA:DNA or heteroduplexes, e.g., RNA:DNA.
  • Complementary - Two sequences are complementary when the sequence of one can bind to the sequence of the other in an anti-parallel sense wherein the
  • 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.
  • 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 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.
  • Receptor any compound or composition capable of recognizing a particular spatial and polar organization of a molecule, e.g., epitopic or determinant site.
  • Illustrative receptors include naturally occurring receptors, e.g., thyroxine binding globulin, antibodies, enzymes, Fab fragments, lectins, nucleic acids, repressors, protection enzymes, protein A, complement component C1q, DNA binding proteins or ligands and the like.
  • 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.
  • inorganic powders such as silica, magnesium sulfate, and alumina
  • natural polymeric materials particularly cellulosic materials and materials derived from cellulose
  • Natural or synthetic assemblies such as liposomes, phospholipid vesicles, and cells can also be employed. Binding of sbp members to a support or surface may be accomplished by well-known techniques, commonly available in the literature. See, for example, "Immobilized Enzymes,” Ichiro Chibata, Halsted Press, New York (1978) and Cuatrecasas, J. Biol. Chem., 245:3059 (1970).
  • the surface can have any one of a number of shapes, such as strip, rod, particle, including bead, and the like.
  • Label a member of a signal producing system.
  • the label is part of an oligonucleotide probe either being conjugated thereto or otherwise bound thereto or associated therewith and is capable of being detected directly or indirectly.
  • Labels include reporter molecules that can be detected directly by virtue of generating a signal, specific binding pair members that may be detected indirectly by subsequent binding to a cognate that contains a reporter molecule, oligonucleotide primers that can provide a template for amplification or ligation or a specific polynucleotide sequence or recognition sequence that can act as a ligand such as for a repressor protein, wherein in the latter two instances the oligonucleotide primer or repressor protein will have, or be capable of having, a reporter molecule.
  • any reporter molecule that is detectable can be used.
  • the reporter molecule can be isotopic or nonisotopic, usually non-isotopic, and can be a catalyst, such as an enzyme, a polynucleotide coding for a catalyst, promoter, dye, fluorescent molecule, chemiluminescer, coenzyme, enzyme substrate, radioactive group, a small organic molecule, amplifiable polynucleotide sequence, a particle such as latex or carbon particle, metal sol, crystallite, liposome, cell, etc., which may or may not be further labeled with a dye, catalyst or other detectable group, and the like.
  • a catalyst such as an enzyme, a polynucleotide coding for a catalyst, promoter, dye, fluorescent molecule, chemiluminescer, coenzyme, enzyme substrate, radioactive group, a small organic molecule, amplifiable polynucleotide sequence, a particle such as latex or carbon particle, metal sol,
  • the reporter group can be a fluorescent group such as fluorescein, a chemiluminescent group such as luminol, a terbium chelator such as N-(hydroxyethyl) ethylenediaminetriacetic acid that is capable of detection by delayed fluorescence, and the like.
  • the label is a member of a signal producing system and can generate a detectable signal either alone or together with other members of the signal producing system.
  • a reporter molecule can be bound directly to a 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 Application Serial No. 07/555,323 filed July 19, 1990, the relevant disclosure of which is incorporated herein by reference.
  • the signal producing system may have one or more components, at least one component being a label.
  • the signal producing system generates a signal that relates to the presence or amount of target polynucleotide in a sample.
  • the signal producing system includes all of the reagents required to produce a measurable signal.
  • the labels and other reagents of the signal producing system must be stable at the temperatures used in an amplification of a target polynucleotide. Detection of the signal will depend upon the nature of the signal producing system utilized.
  • the signal producing system is characterized in that the members of the system are chosen such that the binding of the second and third oligonucleotide probes to their respective duplexes to form termolecular complexes alters the signal generated by the signal producing system of the respective termolecular complexes.
  • the second oligonucleotide probe and the third oligonucleotide probe comprise a member of a different signal producing system and the signals are measured from the ternary complexes and a ratio of signals is determined.
  • the first oligonucleotide probe may comprise a member of both signal producing systems.
  • first oligonucleotide probe in combination with each of the second oligonucleotide probe and the third oligonucleotide probe comprise members of different signal producing systems and the signals measured from the ternary complexes are used to determine a ratio of signals.
  • first oligonucleotide probe comprises a first member of a each of two signal producing systems and the second oligonucleotide probe and the third oligonucleotide probe each respectively comprise a second member of each of the signal producing systems.
  • the first member is a catalyst such as an enzyme and the second members are catalysts such as enzymes that are different from the first enzyme and from each other and the products of the reaction of the enzyme comprising the first member are the substrates for the other of the enzymes.
  • the first member is a catalyst such as an enzyme
  • the second members are catalysts such as enzymes that are different from the first enzyme and from each other and the products of the reaction of the enzyme comprising the first member are the substrates for the other of the enzymes.
  • a list of enzymes is found in U.S. Patent No. 4,299,916 at column 30 to column 33.
  • coupled catalysts usually two or more enzymes, where the product of one enzyme serves as the substrate of the other enzyme.
  • One of the enzymes is used as the label in the first oligonucleotide probe.
  • Different second enzymes are used in the second and third oligonucleotide probes.
  • the solute will be the substrate of any one of the enzymes, but preferably of an enzyme bound to the first oligonucleotide probe.
  • the enzymatic reaction may involve modifying the solute to a product that is the substrate of another enzyme or production of compound that 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 enzyme.
  • a substrate includes coenzymes
  • Medola blue can catalyze the conversion of NAD and hydroquinones to NADH, which reacts with FMN oxidoreductase and bacterial luciferase in the presence of long chain aldehydes to produce light.
  • Examples of particular catalytic systems that may be utilized in the present invention are found in U.S. Patent No. 4,299,916 at column 33, line 34, to column 38, line 32, the disclosure of which is incorporated herein by reference.
  • additional members of the signal producing system include enzyme substrates and so forth.
  • the product of the enzyme reaction is preferably a luminescent product or a fluorescent or non-fluorescent dye, any of which can be detected spectrophotometrically, or a product that can be detected by other spectrometric or electromethc means.
  • the first member of the signal producing system is a quencher and the second members are fluorescent compounds that emit at different wavelengths or with different decay rates.
  • Fluorescers of interest generally emit light at a wavelength above 350 nm, usually above 400 nm and preferably above 450 nm. Desirably, the fluorescers have a high quantum efficiency, a large Stokes shift and are chemically stable under the conditions of their conjugation and use.
  • the term fluorescer is intended to include substances that emit light upon activation and include fluorescent and phosphorescent substances, scintillators and chemiluminescent substances. In this approach the medium is irradiated with light and the fluorescence is determined.
  • Fluorescers of interest fall into a variety of categories having certain primary functionalities.
  • These primary functionalities include 1- and 2-aminonaphthalene, p,p-diaminostilbenes, pyrenes, quaternary phenanthridine salts, 9-aminoacridines, imines, anthracenes, oxacarboxyamine, merocyanine, 3-aminoequilenin, perylene, bis-benzoxazole, bis-p-oxazolyl benzene, 1 ,2-benzophenazine, retinal, bis-3- 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.
  • the quencher is a fluorescent compound or fluorescer but energy acceptors that have weak or no fluorescence are also useful.
  • quenchers are the xanthene dyes, which include the fluoresceins derived from 3,6-dihydroxy-o- phenyl-xanthhydrol and rhodamines, derived form 3,6-diamino-9- phenylxanthhydrol.
  • Another group of compounds are the naphthylamines such as, e.g., 1-anilino-8-naphthalene sulfonate, 1-dimethylaminonaphthyl-5-sulfonate and the like.
  • quenchers that may be employed are those fluorescers of interest mentioned above wherein one fluorescer can absorb the energy of another fluorescer and quench its fluorescence.
  • Energy acceptors that are non-fluorescent can include any of a wide variety of azo dyes, cyanine dyes, 4,5-dimethoxyfluorescein, formazans, indophenols and the like.
  • quenchers is energy absorbent or quenching particles.
  • examples of such particles are carbon particles, such as charcoal, lampblack, graphite, colloidal carbon and the like.
  • carbon particles metal sols may also find use, particularly of the noble metals, gold, silver, and platinum.
  • Other metal derived particles may include metal sulfides, such as lead, silver or copper sulfides or metal oxides, such as iron or copper oxide.
  • Heller U.S. Patent No. 5,565,322 discloses donor and acceptor chromophores at column 9, line 37, to column 14, line 7, the disclosure of which is incorporated herein by reference.
  • fluorescers and quenchers may also be found in U.S. Patent Nos. 4,261 ,968, 4,174,384, 4,199,983 and 3,996,345, the relevant disclosures of which are incorporated herein by reference.
  • the first member of the signal producing system is a sensitizer and the second members are chemiluminescent compounds that emit at different wavelengths or with different decay rates.
  • the first member is a chemiluminescent compound and the second members are sensitizers that can be independently excited by different wavelengths of light.
  • chemiluminescent compounds and sensitizers are set forth in U.S. Patent No. 5,709,994, the disclosure of which is incorporated herein by reference.
  • 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.
  • these compounds react with singlet oxygen to form dioxetanes or dioxetanones.
  • the latter are usually electron rich olefins.
  • 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.
  • LOCI - Luminescent Oxygen Channeling Immunoassay The signal producing system (Dade Behring, Inc., Deerfield, Illinois) may be used homogeneously or heterogeneously in all types of assays, including but not limited to, immunoassays. Reagents and methods for LOCI are more fully described herein and in Ulman, E.F. et al., Clin. Chem. (1996) 42:9, pp. 1518-1526.
  • Termolecular complex - a complex formed in accordance with the present methods upon the binding of the two oligonucleotide probes with a target polynucleotide or a reference polynucleotide.
  • Such complex is termolecular in that it involves three molecules, namely, the two oligonucleotide probes and the single strand of such target polynucleotide or reference polynucleotide.
  • the termolecular complex is relatively stable under the isothermal conditions employed.
  • body fluids such as, for example, whole blood, blood fractions such as serum and plasma, synovial fluid, cerebro- spinal fluid, amniotic fluid, semen, cervical mucus, sputum, saliva, gingival fluid, urine, and the like.
  • the amount of the sample depends on the nature of the sample and the analyte contained therein. For fluid samples such as whole blood, saliva, urine and the like the amount of the sample is usually about 1 to 1000 microliters, more usually, about 10 to 100 microliter.
  • the sample can be pretreated and can be prepared in any convenient medium, which does not interfere with the reactions conducted
  • buffers will normally be present in the assay medium, as well as stabilizers for the assay medium and the assay components.
  • proteins may be included, such as albumins, organic solvents such as formamide, quaternary ammonium salts, polycations such as dextran s ⁇ lfate, surfactants, particularly non-ionic surfactants, binding enhancers, e.g., polyalkylene glycols, or the like.
  • One embodiment of the present invention is directed to a method for detecting one or two polynucleotide analytes.
  • the polynucleotide analyte(s) may be one or both a reference (wild type) polynucleotide and a target (mutant) polynucleotide.
  • the target and reference polynucleotides are single stranded either in a natural state or rendered single stranded as described above.
  • a sample suspected of containing the polynucleotide analyte(s) is combined in a medium with at least two oligonucleotide probes.
  • the probes include a polynucleotide sequence which fully hybridizes with the sequences of the target or reference polynucleotides, respectively. "Fully hybridizes or fully complimentary" means that the sequences are exactly complementary.
  • the medium is subjected to conditions substantially irreversibly hybridizing the probes to the target and reference sequences. If only one of the target and reference polynucleotides is present in the medium, the probes will compete for hybridization to the sequence present.
  • the probe that is fully complementary to the sequence present will substantially inhibit the non- complementary probe from hybridizing, even if the non-complementary probe sequence is mismatched by only one nucleotide.
  • the probe which is fully complementary polynucleotide, has a label that can be distinguished from a label on the probe that is fully complementary to the reference polynucleotide.
  • the hybridization of one the probes to either the target or reference polynucleotide can be detected.
  • two signals can be detected.
  • a third probe known as a common probe is combined in the reaction medium.
  • the common probe substantially irreversibly hybridizes to both the reference polynucleotide and the target polynucleotide.
  • the common probe is labeled with a first member of a signal producing systems when the target and reference probes are labeled with second members of a signal producing systems.
  • the signal produced by the interaction of the labels on the common probe and the reference probe is different than the signal produced by the interaction of the labels on the common probe and the target probe.
  • Reference polynucleotide WT has a sequence CS that is common with sequence CS of the target polynucleotide Mu.
  • a common oligonucleotide probe CP has a sequence PCS that is hybridizable with sequence CS of WT and Mu.
  • WT and Mu both contain second sequences, WTS and MuS, respectively, which are identical except for the difference of one nucleotide SNP.
  • a second oligonucleotide probe WTP contains a sequence WTPS fully complementary to sequence WTS.
  • a third oligonucleotide probe MuP contains a sequence MuPS identical to WTP except for a difference of one nucleotide cSNP, which is complementary to SNP. Therefore, MuP is fully complementary to MuS.
  • All of the polynucleotides and oligonucleotide probes are combined in an appropriate medium and subjected to conditions for allowing hybridization of the probes to WT and Mu. After an appropriate period for complex formation, signals are determined from the reaction medium. One signal results from the interaction of label L1 on CP and label L2 on WTP. A second signal results from the interaction of label L1 on CP and label L3 on MuP. Labels L1 and L2 are part of a signal producing system as discussed above.
  • L1 and L2 can interact to produce a signal that is then measured.
  • L1 and L3 can interact to produce a signal that can be measured.
  • the signal produced by the interaction of L1 and L2 is different from the signal produced by the interaction of L1 and L3.
  • the labels can be part of the probes initially.
  • the labels can be included as separate reagents either in the initial reaction mixture or added subsequent to the incubation period.
  • an sbp member may be attached to CP and the corresponding sbp member attached to label L1.
  • the two sbp members bind to each other and L1 becomes part of the common probe CP.
  • the two sbp members may be, for example, hapten-antibody, nucleic acid-nucleic acid, and the like.
  • Labels L2 and L3 may be incorporated with WTP and MuP in a similar manner.
  • the labels L1 , L2 and L3 include oligonucleotide sequences L1S, L2S and L3S respectively, which are complementary to the sequences on the probes which are not complementary to the target or reference sequences.
  • L1 includes a sequence L1S capable of hybridizing sequence cL1S of the common probe CP.
  • L2 includes a sequence L2S capable of hybridizing sequence cL2S on the wild type probe WTP.
  • L3 includes a sequence L3S capable of hybridizing to sequence cL3S on the Mutant Probe MuP.
  • the labels when combined in the reaction medium with the probes and target and reference sequences, will bind to the probes.
  • the labels can be manufactured independent of the probes.
  • the labels can be used with any probe so long as the probe includes a tail that can bind the probe molecule.
  • the tail can be as simple as a poly-A or poly-T tail. This feature allows for simple and cost effective manufacture of probes since the probes added to the reaction medium of the present invention are unlabeled.
  • the probes include both a sequence that hybridizes to a label and a sequence which hybridizes to a complementary sequence on the target or reference polynucleotides.
  • the probe sequence that hybridizes to a complementary sequence on the target or reference polynucleotide is ussually about 5-80 nucleotides, preferably about 10 to about 50 nucleotides, more preferably about 15 to about 30 nucleotides, in length.
  • the probe sequence which is specific for the target sequence MuP should be fully complementary to the target sequence MuS. Consequently, it is preferred that the probe sequence MuP is 100% complementary to the target sequence MuS.
  • the probe sequence specific for the reference sequence WTP and the reference sequence WTS are 100% complementary..
  • the degree to which the sequences must be complementary depends on the relative lengths and nucleotide composition of the sequence of the oligonucleotide probes.
  • An important feature of the invention is that the probe specific for the reference sequence WTP and the probe specific for the target sequence MuP compete for binding to the target or reference sequences present in the sample. To the extent that longer probes may be less than 100% complementary to their respective sequences, in addition to the difference of a SNP or mutation, depends on the ability of the probe sequence which is most complementary to the target sequence to inhibit the binding of the less complementary probe sequence to the target. It is contemplated as part of the invention that probes which are less than 100% complementary to a respective target or reference sequence will bind the target or reference sequence preferentially over a less complementary probe.
  • the competition may be established by controlling reactions conditions including time, temperature and speed of lowering the temperature of the reaction medium to allow for hybridization conditions.
  • the sequence of the common probe which binds both the reference and target sequence CPS need not be fully complementary. However, the common probe should substantially irreversibly bind to the target and references above the temperature at which the target specific probe and reference specific probe substantially bind to the target and reference, respectively.
  • more than two polynucleotide sequences, representing multiple alleles for a gene may be detected. Alleles that occur in more than two variants may be detected by employing additional nucleotide probes for which a signal can be detected.
  • the additional probes would be identical to the first and second probes described above except for the difference of one or nucleotides representing the difference As long as the label for each additional probe can be distinguished, the allelle corresponding complementary sequence of each probe can be detected.
  • complementary probes specific for each allele sequence can be employed. The probes will compete with each other for binding to the sequence to which they are most complementary. Further, the probes which are most complementary to any particular sequence will substantially inhibit any less complementary probe from binding.
  • the length of the various oligonucleotide probes and the relevant sequences contained therein may be greater or less than that indicated above. The length depends on a number of factors such as the possibility of strand invasion, whether the nucleotides are natural or modified, the temperature, the pH, the salt concentration of the medium, and so forth. In general, the length of the above sequences should be sufficient to ensure substantially non-dissociative complexes.
  • an aqueous medium is usually employed. In general, an aqueous medium is employed for the entire method in accordance with the present invention.
  • polar cosolvents may also 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.
  • the pH and temperature are chosen based on the particular method of amplification employed.
  • the present invention can be used for the direct detection of nucleic acid in quantities as little as 10 ⁇ 12 or less without amplification.
  • the invention may be carried out with amplification of the target and references sequences. Any amplification method may be employed.
  • 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 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 spectromet c or electrometric means.
  • the reporter molecule is a fluorescent molecule
  • the medium can be irradiated and the fluorescence determined.
  • the label is a radioactive group
  • the first probe can be separated from the duplex of the first and second probes and one of the fractions can be counted to determine the radioactive count.
  • the association of the labels within the termolecular complex may also be determined by using labels that provide a signal only if the labels become part of, or dissociate from, the complex.
  • the binding of the single stranded target polynucleotide, if present, to the second probe causes displacement of the first probe from the second probe and thereby alters a signal generated by the signal producing system. This approach is particularly attractive when it is desired to conduct the present invention in a homogeneous manner.
  • Such systems include enzyme channeling immunoassay, fluorescence energy transfer immunoassay, electrochemiluminescence assay, induced luminescence assay, latex agglutination and the like.
  • detection of the complex is accom- pushed by employing at least one suspendable particle as a support, which may be bound directly to a nucleic acid strand or may be bound to an sbp member that is complementary to an sbp member attached to a nucleic acid strand, either first or second oligonucleotide probe.
  • a suspendable particle 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 which is 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. Patent No. 5,709.994, the disclosure of which is incorporated herein by reference), chemiluminescent and electroluminescent la- bels.
  • 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.
  • the particles may be simple latex particles or may be particles comprising a sensitizer, chemiluminescer, fluorescer, dye, and the like.
  • Typical particle/reporter molecule pairs include a dye crystallite and a fluorescent label where binding causes fluorescence quenching or a tritiated reporter molecule and a particle containing a scintillator.
  • Typical reporter molecule pairs include a fluorescent energy donor and a fluorescent acceptor dye.
  • Typical particle pairs include (1 ) two latex particles, the association of which is detected by light scattering or turbidimetry, (2) one particle capable of absorbing light and a second label particle which fluoresces upon accepting energy from the first, and (3) one particle incorporating a sensitizer and a second particle incorporating a chemiluminescer as described for the induced luminescence immunoassay referred to in U.S. Serial No. 07/704,569, filed May 22, 1991 , entitled “Assay Method Utilizing Induced Luminescence", which disclosure is incorporated herein by reference.
  • detection of the termolecular complex using the induced luminescence assay as applied in the present invention involves employing a photosensitizer as part of one label and a chemiluminescent compound as part of the other label. If the complex is present the photosensitizer and the chemiluminescent compound come into close proximity. The photosensitizer generates singlet oxygen and activates the chemiluminescent compound when the two labels are in close proximity. The activated chemiluminescent compound subsequently produces light. The amount of light produced is related to the amount of the complex formed. If the amplification uses a DNA polymerase, preferably, the oligonucleotide probes are blocked at the 3'-end to avoid any potential interference with and during amplification.
  • Blocking may be accomplished, for example, by employing a group that cannot undergo chain extension, such as, for example, an unnatural group such as a 3'-phosphate, a 3'-terminal dideoxy, an abasic ribophosphate, a polymer or surface, or other means for inhibiting chain extension.
  • a polynucleotide that does not hybridize to the amplicon is attached to the 3'-end.
  • Such an end group can be introduced at the 3' end during solid phase synthesis or a group can be introduced that can subsequently be modified.
  • a ribonucleotide 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.
  • reagents employed in the present invention can be provided in a kit in package combination.
  • the kit comprises in package combination one or more reagents for conducting amplification and detection of a polynucleotide.
  • kits in accordance with the present invention is a kit comprising a first oligonucleotide probe having a sequence which hybridizes to a first region of a target polynucleotide in a sample, a second oligonucleotide probe which hybridizes to a second region of the target polynucleotide and a third oligonucleotide probe having the same sequence as the sequence of the second probe which hybridizes to the second region of the target polynucleotide except for the difference of one or more nucleotides or the insertion or deletion of one or more nucleotides.
  • the difference, insertion or deletion being complimentary to an expected mutation of the target polynucleotide.
  • the kit further comprises a first label having a first member of first and second signal producing systems, a second label comprising a second member of a second signal producing system and a third label comprising a second member of a second signal producing system.
  • the first, second and third labels are capable of noncovalently binding to the first, second and third probes, respectively.
  • the first member of the signal producing system may be a sensitizer and the other members being chemiluminescers.
  • the first member of the signal producing system may be a chemiluminescer and the second member of the signal producing system may be a sensitizer. Signals from the respective labels 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 reagents.
  • the kit above can further include in package combination reagents for conducting an application of the target polynucleotide.
  • the kit may contain forward and reverse primers, a polymerase, and dNTP's.
  • the relative amounts of the various reagents in the kit can be varied widely to provide for concentrations of reagents necessary to achieve the objectives of the present invention.
  • one or more of the reagents in the kit can be provided as a dry powder, usually lyophilized, including excipients which on dissolution will provide for 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 kit may also include a written description of a method in accordance with the present invention as described above.
  • UV-visible absorption spectra were done on a HP 8452A diode array spectrophotometer. Fluorescence and chemiluminescence measurements were done on a Spex fluorolog spectrophotometer or a Perkin Elmer 650-40 spectrophotometer. Chemiluminescence measurements were also performed on an in-house chemiluminometer. Toluene was distilled from sodium over argon. Unless mentioned otherwise, all solvents were used without purification, and most reactions were carried out under argon. Silica gel used for flash chromatography was 230-400mesh ASTM, purchased from Scientific Products while preparative plates (1000 ⁇ ) and analytical plates were purchased from Analtech.
  • Silicon phthalocyanine and silicon naphthalocyaninine were prepared as described below and phthalocyanine tetrasulfonates was obtained from Ultra Diagnostics, Inc.
  • Carboxylate-modified polystyrene (latex) particles were purchased from Seradyn, Inc. The particles were 203 ⁇ 4.0 nM.
  • the carboxyl parking area was 49.5 angstroms squared (0.09 milliequivalents/g). Solids were 10% (100 mg/ml).
  • 2-ethoxyethanol was from Aldrich Chemical Co. and was redistilled under vacuum.
  • Sodium hydroxide was 0.1 N.
  • Isopropanol was from Aldrich Chemical Co..
  • 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 HCl - Tris(hydroxymethyl)aminomethane-HCI (a 10X solution) from BioWhittaker, Walkersville, Maryland
  • 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 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 H 2 0 (2x), brine and were dried over MgS0 4 .
  • 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 high vacuum (oil pump at about 1 mm Hg) to dryness.
  • the 6-t-butyl-2,3-dicyanonaphthalene (I) (9.07 g, 38.7 mmol) was suspended in 50 mL MeOH. While stirring under argon, 8.5 mL of 1.05 N NaOMe/MeOH (freshly prepared from Na°) was added and the light yellow suspension was bubbled with NH 3 gas for 1 h at ambient temperature. Dilution with an additional 75 ml MeOH did not dissolve the fine suspension. Bubbling with NH 3 was continued at 65° for 2 h until the suspension cleared, and then an additional 1 h. Volatiles were removed by rotary evaporation, and the sticky semi-solid was triturated with 200 mL water.
  • the 6-t-Butyl-diiminobenzisoindoline (2) (9.01 g, 35.9 mmol). Prepared as described above, was stirred in 100 mL quinoline. Silicon tetrachloride (4.0 mL, 35 mmol) was added in 1 mL increments at such a rate (-30 min) that the mild exotherm was maintained below 30°. The reaction was then gradually heated by oil bath from 60° to 180°, and stirred under argon for 1 h at that temperature. After cooling to ambient temperature, the reaction was pipetted into two 250 cc polypropylene centrifuge bottles, each containing 150 mL 1 :1 H 2 0/MeOH.
  • the deep green suspension was gradually heated to 100°C After 30 min, frothing subsided, and heating was continued for two hours. After cooling, the suspension was rinsed with 200 mL water into two 250 cc centrifuge bottles. The solid was washed repeatedly by centrifugation (3x, 6K to 12 K rpm) with 150 mL portions of water/bottle. Because of poor compaction, a final centrifugation was done in two 30 cc tubes with 25 mL portions of water at 16 K rpm. The solid was vacuum dried (16 h, 30 mmHg, 60°C, P 2 0 5 ) and weighed (2.822 g).
  • the t-bu -Nc(OH) 2 (4) (1.61 g, 1.6 mmol), prepared as described above, was dissolved in a solution of 15 mL tri-n-hexylsilylchloride / 30 mL dry pyhdine. After bubbling with argon, the solution was heated at 120° for 1 h. Heating the reaction for an additional 2 h did not alter the product distribution as judged by TLC (Si0 2 , 10% CH 2 CI 2 /hexane). The reaction was cooled to ambient temperature, and the reaction mixture was partitioned between 150 mL hexane and 100 mL water. The mixture was stirred for 30 min until the hexane phase cleared to a deep green color.
  • the aqueous phase was removed and the organic phase was washed twice with 150 mL portions of water, until the final aqueous phase was colorless (pH about 6).
  • the organic phase was filtered (glasswool/funnel), concentrated to about 25 mL, and chromatographed (Si0 2 ; 5% CH 2 CI 2 /hexane). Re-chromatography (Chromatotron: Si0 2 rotor, 0 to 10% CH 2 CI 2 /hexane) was necessary to separate the t-butyl isomers. A portion of the major isomer (94 mg) was isolated and used as is for bead dyeing.
  • N-(2,3-epoxypropyl)phthalimide The mixture was heated and stirred in a 90°C water bath for two hours. A small aliquot was precipitated three times from methanol and analyzed by NMR. The appearance of a peak at 7.3-7.6 indicated incorporation of phthalimide. The main reaction mixture was precipitated by addition to 3.5 L of methanol, after which solid was collected. The phthalimide protecting group was removed by dissolving the product above in 500 mL of 0.1 M acetate buffer, adding 50 mL of 35% hydrazine, and adjusting the pH to 3.5.
  • the mixture was heated at 80°C for 1 h, the pH was re-adjusted to 3.2, and the mixture was heated for an additional half hour. An aliquot was precipitated three times in methanol. NMR showed that the phthalimide group was no longer present.
  • the reaction mixture was neutralized to pH 8 and stored at room temperature.
  • the product was purified by tangential flow filtration using a 50,000 molecular weight cut-off filter, washing with water, 0.01 M HCl, 0.01 M NaOH, and finally water.
  • the product solution was concentrated by filtration to 700 mL then lyophilized. Determination of reactive amines using trinitrobenzenesulfonate gave about 1 amine per 16 glucose residues.
  • the sensitizer beads were prepared by placing 600 mL of carboxylate modified latex (Seradyn) in a three-necked, round-bottom flask equipped with a mechanical stirrer, a glass stopper with a thermometer attached to it in one neck, and a funnel in the opposite neck.
  • the flask had been immersed in an oil bath maintained at 94 ⁇ 1°C
  • the beads were added to the flask through the funnel in the neck and the bead container was rinsed with 830 mL of ethoxyethanol, 1700 mL of ethylene glycol and 60 mL of 0.1 N NaOH and the rinse was added to the flask through the funnel.
  • the funnel was replaced with a 24-40 rubber septum.
  • the beads were stirred at 765 rpm at a temperature of 94 ⁇ 1 °C for 40 min.
  • Silicon tetra-t-butyl phthalocyanine (10.0 g) was dissolved in 300 mL of benzyl alcohol at 60+/-5°C Eighty-five (85) mL was added to the above round bottom flask through the septum by means of a 100 ml syringe heated to 120+/- 10°C at a rate of 3 mL per min. The remaining phthalocyanine solution was then added similarly. The syringe and flask originally containing the phthalocyanine was rinsed with 40 mL of benzyl alcohol and transferred to a round-bottom flask. After 15 min 900 mL of deionized water and 75 mL of 0.1 N NaOH was added dropwise over 40 min.
  • the temperature of the oil bath was allowed to drop slowly to 40+/- 10°C and stirring was then discontinued.
  • the beads were then filtered through a 43 micron polyester filter and subjected to a Microgon tangential flow filtration apparatus (Microgon Inc., Madison Hills, CA) using ethano water, 100:0 to 10:90, and then filtered through a 43 micron polyester filter.
  • Hydroxypropylaminodextran solution was prepared at 2 mg/mL in 50 mM MES pH 6.
  • One hundred fifty mg phthalocyanine sensitizer beads in 7.5 mL water was added dropwise to 7.5 mL of the hydroxypropylaminodextran solution while vortexing.
  • One hundred eighty eight ⁇ l of EDAC solution (80 mg/ml) in water was added to the coating mixture while vortexing. The mixture was incubated overnight at room temperature in the dark. The mixture was diluted with 12 mL water and centrifuged. The supernatant was discarded and the bead pellet suspended in 40 mL water by sonication.
  • the beads were washed 3 times with water (40 ml per wash) by repeated centrifugation and suspension by sonication. The final pellet was suspended in 5 mL water.
  • naphthalocyanine (Nc) dyed sensitizer particles A 10 % suspension of carboxylated latex beads (4.4 ml) was mixed with 4.4 mL ethoxyethanol, 8.8 mL ethylene glycol, and 0.44 mL 0.1 N sodium hydroxide solution. Naphthalocyanine (0.475 mg) was dissolved in 0.4 mL benzyl alcohol.
  • the suspension was centrifuged at a slow speed to pellet a trace of debris from the dyeing procedure.
  • the beads remaining in the supernatant were decanted and stored at 4 degrees C Hydroxypropylaminodextran solution was prepared at 10 mg/mL in 50 mM
  • MES pH 6 Twenty mg of the naphthalocyanine sensitizer beads in 1 mL 10 % ethanol in water was slowly added to 1 mL of the hydroxypropylaminodextran solution while vortexing. Two mg EDAC dissolved in 0.2 mL water was added to the coating mixture while vortexing. Following an overnight incubation at room temperature, the mixture was centrifuged, the supernatant discarded, and the bead pellet suspended in 2 mL water by sonication. The water wash was repeated 2 times by centrifugation and the final bead pellet suspended in 1 mL water.
  • the dye solution was poured into the bead suspension and stirred for 20 min at 95°C, then allowed to cool slowly to about 47°C and filtered through a 43 ⁇ m polyester filter to remove any debris generated during the dyeing procedure.
  • the beads were washed by tangential flow filtration using a Microgon apparatus with a P698/4 filter. After priming of the system with wash solvent (1 :2 v/v ethoxyethanol and ethylene glycol), the dyed bead mixture was added, concentrated to about 20 mg/mL, then washed with 6 liters of wash solvent and 4.8 L of 10% v/v ethanol in water adjusted to pH 10 with NaOH.
  • the TAR beads were concentrated to about 50 mg/mL during the wash, then stored at 4 °C protected from light.
  • a plasticizer was incorporated into the beads to enhance the rate of decay of luminescence.
  • a mixture was prepared containing 250 ⁇ L of n- heptadecylbenzene, 20 mL of ethanol, and 0.5 g of hydroxypropylaminodextran dissolved in 25 mL of 50 mM MES pH 6. The mixture was heated to 80°C in an oil bath and stirred vigorously to disperse the plasticizer.
  • a second mixture containing 40 mL of dyed TAR beads from above (diluted to 25 mg/mL in 10% ethanol) and 30 mL of 50 mM MES pH 6 was also heated to 80°C The two mixtures were combined and left stirring at 80°C overnight.
  • the beads were separated, by pipette, from a small amount of excess plasticizer that floated on top of the bead suspension.
  • EDAC 200 mg
  • 3 mL of water was added, and the mixture was stirred at room temperature for 2 h.
  • the mixture was then centrifuged to recover the beads.
  • the bead pellet was suspended by sonication and washed with three 40 mL portions of water by alternating centrifugation and suspension by sonication.
  • the final bead pellet was then suspended in about 35 mL water.
  • hydroxypropylaminodextran coated phthalocyanine sensitizer beads were suspended in 5 mL 50 mM MOPS pH 7.
  • a 10 % (w/v) SIAX solution was prepared in DMF and 77 ⁇ l added to the bead suspension while vortexing. The mixture was incubated at room temperature for 90 min in the dark and then a second 77 ⁇ l aliquot of SIAX solution added and the mixture incubated for an additional 60 min. The suspension was centrifuged and the supernatant discarded. The bead pellet was suspended in 6 mL water by sonication and the centrifugation repeated.
  • Coupling buffer consists of the following mixture: (900 ⁇ L 0.2 M borate, 2 mM EDTA pH 9 and 333 ⁇ L of 0.4 M borate pH 9.45 and 1000 ⁇ L of 2 M Na 2 S0 4 ) which had been degassed and saturated with argon.
  • Tween 20 detergent was added to the coupling buffer mixture after degassing and saturating with argon.
  • oligonucleotide solution Seven hundred sixty ⁇ L of oligonucleotide solution was placed in a centrifuge tube and 76 ⁇ L of 2.5 M NaOAc pH 5.3 added. One hundred forty seven ⁇ L of 20 mM TCEP in water was added to the oligonucleotide solution and the mixture incubated 1 h at room temperature in the dark. Four volumes of 200 proof ethanol was added to the mixture to precipitate the reduced oligonucleotide. Precipitation was facilitated by placing the mixture in a -20 degree C freezer for 1 hour. The precipitate was collected by centrifugation and then dissolved in 495 ⁇ L 5 mM Na 2 HP0 4 , 2 mM EDTA pH 6 that had been degassed and saturated with argon.
  • the oligonucleotide solution was then added to the bead pellet under coupling buffer and the mixture was sonicated to suspend the beads.
  • the suspension was incubated at 37 degrees C for 23 h.
  • Residual iodo groups on the iodoaminodextran coat were capped by reaction with mercaptoacetic acid.
  • the bead suspension was centrifuged and the supernatant removed.
  • the pellet was suspended by sonication in 5 mL of 10 mM mercaptoacetic acid in 0.4 M borate pH 9.45 and the mixture incubated at 37 degrees C for 1 h.
  • the beads were recovered by centrifugation and suspended in 5 mL blocking buffer: (0.1 M NaCl, 0.17 M glycine, 10 mg/mL BSA, 0.1 % Tween 20, 1 mM EDTA pH 9.2 sterile filtered and 50 ⁇ L/mL Calf Thymus DNA added).
  • the mixture was incubated for 3 h at 37 degrees C Following centrifugation, the beads were washed twice by centrifugation with 5 mL Buffer A per wash. The final pellet was suspended in 6 mL IHBB buffer and incubated at 95 degrees C for 90 min. After cooling, the beads were centrifuged, the supernatant was discarded, and the pellet was suspended in 6 ml of equal volumes of 0.125 M NaOAc pH 5 and 30% hydrogen peroxide solution. Incubate at 37 degrees for 2.5 hours.
  • the mixture was centrifuged, the supernatant discarded, and the beads washed 3 times by centrifugation with storage buffer (50 mM KCl, 10 mM tris, 4 mM EDTA, 0.2% acetylated BSA pH 8.2) using 5 mL buffer per wash.
  • storage buffer 50 mM KCl, 10 mM tris, 4 mM EDTA, 0.2% acetylated BSA pH 8.2
  • the final pellet was suspended by sonication in 6 mL storage buffer and stored at 4 degrees protected from light.
  • (ATAG)g oligonucleotide coated naphthalocyanine sensitizer particles Nc-(ATAGW)
  • the oligonucleotide coated beads were prepared by a procedure similar to that described above for the preparation of Pc-A 2 beads.
  • oligonucleotide coated beads were prepared by a procedure similar to that described above for the preparation of Pc-A 2 beads. However, these particles were not treated with peroxide.
  • Standard PCR based protocols were used to convert 70 base single stranded synthetic WT and Mu oligonucleotides into 130 bp double stranded amplicons.
  • the following sequences derived from the S. aureus mecR1 gene (Genbank Accession No. X63598) were chemically synthesized by Oligos Etc, Wilsonville, Oregon:
  • GO-1 reverse linking primer for primer for WT and Mu oligonucleotides
  • GO-2 forward linking primer for WT oligonucleotide
  • FC-1 forward primer
  • FC-2 reverse primer 5' - TAGAATAAGCTTGCTCCCGTTCAT (SEQ ID NO: 10)
  • Primer extension were performed in 100 ⁇ l volumes in 10 mM Tris-HCI (pH 8.4), 50 mM KCl, 0.2 mg/ml BSA, 4 mM MgCI 2 0.2 mM each dATP, dCTP, dTTP, dGTP, and 5 units cPfu (Stratagene).
  • the 20 nM single stranded Mu and WT DNA was converted into double stranded amplicons by primer extension with 20 nM GO-1. The reaction was heated to 65°C for 3 minutes at which point the polymerase was introduced to prevent mis-priming.
  • the primer annealing and extension was carried out at 50°C (15 minutes), 60°C (10 minutes .), and 73°C (5 minutes).
  • the double stranded DNA produced was further increased in size by using specific primer GO-2 for WT and primer GT for Mu using similar reaction conditions except the initial denaturation was carried out at 95 C
  • the single base difference between the original WT and Mu oligonucleotides was maintained during all the primer extension and DNA amplification steps.
  • PCR was subsequently carried out with outer primers FC-1 and FC-2 using the same reaction conditions mentioned above except that the primers were present at 250 nM.
  • the thermal cycling profile was as follows: initial denaturation of double stranded DNA 95°C (3 minutes) followed by 4 cycles of 95°C (20 seconds), 41 °C (1 minute), and 73° (1 minute). The annealing temperature was then increased from 41 °C to 63°C and amplification was continued for 16 more cycles. The temperature was finally held at 75°C for 5 minutes. The product was analyzed on a 4-20% non-denaturating acrylamide gel (Novex Corporation).
  • Detection of 100 pM WT and Mu synthetic amplicons was carried out using a LOCITM assay. Reactions were carried out in 40 ⁇ l reactions containing 10 mM Tris-HCI (pH 8.4), 50 mM KCl, 0.2 mg/ml BSA, 4 mM MgCI2, 0.2 mM each dATP, dCTP, dTTP, dGTP. The dNTPs were added to ensure a consistent reaction medium with the PCR amplification of Example 1.
  • the reaction also contained the 1.0 ug of chemiluminescer particle (dopTAR-(A) 20 ) and 1.0 ug of each of the sensitizer particles (Pc-(AGTA) 6 and Nc-(ATAG) 6 ).
  • Each of the three probes, namely FC-3 (the common probe) and specific probes ED-3 and ED-4 were added at 12.5 nM each.
  • the actual for denaturation and annealing of the probes to the Mu and WT amplicons and LOCITM particles were 95°C for 3 minutes, 50° C for 15 minutes, and 41 ° for 60 minutes.
  • the common probe FC-3 binds to both the WT and Mu amplicon and to the dopTAR chemiluminescer particle (dopTAR-(A) 20 ) via the T 20 probe tail on the probe.
  • the WT specific probe (ED-3) binds to the WT amplicon and to the Pc sensitizer particle (Pc-(AGTA)e) via the (TACT) 5 tail.
  • the Mu specific probe binds specifically to Mu amplicon and to the Nc Sensitizer particle (Nc-(ATAG) ⁇ ) via the (CTAT) 5 tail.
  • the chemiluminescent signal generated by the particle pairs formed was read in Relative Light Units (RLU) by an in-house built LOCITM reader.
  • RLU Relative Light Units
  • the WT signal was obtained first by illuminating the reaction tubes with 675-nm laser (to generate singlet oxygen from Pc) for 1.0 second followed by reading the chemiluminescent signal (RLU) for 1 second. This was followed by a 10-second delay to allow the entire signal from the chemiluminscer to decay completely.
  • the Mu signal (RLU) was then obained by illuminating the tubes with a 780-nm laser (to generate singlet oxygen from Nc) for one second.
  • the Mu chemiluminscent signal was read six times after each of a one-second illumination followed by a one second read. The counts from each of the six reads are totaled.
  • the background (no target) signal is subtracted from the both the WT and Mu signals. After removing the cross talk signal the corrected signals are shown is Table 1.
  • Corrected Pc signal (WT) [S-B Pc minus (S-B Nc x 0.0026)] wherein S-B is Signal Background.
  • the correction factor 0.0026 reflects 0.26% of Nc crossover signal into the Pc signal channel.
  • Amplification of the WT and Mu sequences was performed in 40 ⁇ l reaction containing PCR reagents and 250 nM primers FC-1 and FC-2, 0.4 ug Chemiluminescer particle, 1.6 ug each of the two sensitizer particles (Nc and Pc), 10 nM common probe FC-3 and 30 nM each of the two specific probes ED-3 and ED-4.
  • the thermal cycling profile was as follows: Initial denaturation of double stranded DNA 95°C (3minutes) followed by 33 cycles of
  • the WT and Mu signals shown are corrected signals determined as for
  • CTR cystic fibrosis transmembrane conductance regulator
  • the DNA samples were amplified with PCR using the following primers: CF-1 (forward primer)
  • GGC TCCATATTCAATCGGTTAG SEQ ID NO: 15
  • Amplification of 0.8 ng human DNA samples was performed in a 40 ⁇ l reaction using 250 nM of primers CF-1 and CF-10.
  • PCR reagents included 10 mM Tris-HCI (pH 8.4), 50 mM KCl, 0.2 mg/ml BSA, 4 mM MgCI2, 0.2 mM each dATP, dCTP, dTTP, dGTP, & 2 units cPfu (Statagene).
  • the thermal cycling profile was as follows: Initial denaturation of double stranded DNA at 95°C (3 minutes) followed by 33 cycles of 95°C (20 seconds), 64°C (1 minute), and 73°(1 min) before a final 75°C (5 minutes) step.
  • the amplicons were diluted 1 :40 and 1 :400 in water before being detected with the probes and LOCI particles described herein.
  • the probes used for detection were:
  • LOCI Detection of amplified and diluted product by LOCI was carried out as follows. LOCI reactions were carried out in 40 ⁇ lreactions containing 10 mM Tris- HCI (pH 8.4), 50 mM KCl, 0.2 mg/ml BSA, 4 mM MgCI2, 0.2 mM each dATP, dCTP, dTTP, dGTP. In addition the reaction also contained the 1.0 ug of the dopTAR chemiluminescer particle (dopTAR-(A) 24 ) and 1.0 ug of each of the sensitizer particles (Pc-(AGTA) ⁇ and Nc-(ATAG) 6 .
  • Each of the three probes namely AE-5 (common probe) and specific probes AE-3 and AE-4 were added at 12.5 nM each.
  • the conditions for denaturation and annealing of the probes to the target and LOCI particles were 95°C (3 minutes), 50°(15 minutes), and 41 °(60 minutes).
  • the common probe AE-5 binds to both the WT and Mu amplicons and to the chemiluminescent particle via the probe tail T 2 .
  • the specific probe AE-3 binds to WT amplicon and to Pc sensitizer particle (Pc-(AGTA) 6 via the probe tail (TACT) 5 .
  • the Mu probe binds specifically to Mu amplicon (delta F508) and to the Nc Sensitzer particle (Nc-ATAG) ⁇ via the probe tail (CTAT) 5 .
  • the chemiluminescent signal generated by the particle pairs was read in the in-house built LOCI reader. Signals were read as in Example 1 and the corrected LOCI signal obtained from the final dilutions of amplified DNA is shown in Table 3.
  • the WT and Mu signals for Table 3 are corrected signals determined as for
  • Template-directed dye-terminator incorporation (TDI) assay A homogenous DNA diagnostic method based on fluorescence resonance energy transfer. Proc. Natl. Acad. Sci. 94: 10756-10761.

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Abstract

L'invention concerne un procédé de détection de séquences polynucléotidiques spécifiques, de différences entre des séquences polynucléotidiques, ou de mutations dans de l'ADN génomique. Un échantillon contenant un polynucléotide est combiné avec trois sondes oligonucléotidiques. La première sonde s'hybride avec une première région du polynucléotide. La deuxième et la troisième sonde oligonucléotidiques sont capables de s'hybrider avec une seconde région du polynucléotide. Les séquences de la deuxième et de la troisième sonde qui s'hybrident avec le polynucléotide sont identiques, mis à part une différence d'un ou de plusieurs nucléotides, ou l'insertion ou les délétions d'un ou de plusieurs nucléotides. La différence représente une différence entre un polynucléotide sauvage et un polynucléotide mutant. La première sonde s'hybride de façon irréversible avec le polynucléotide. La deuxième et la troisième sonde sont en concurrence pour s'hybrider avec le polynucléotide, la sonde présentant la séquence la plus complémentaire au polynucléotide inhibant la fixation de l'autre sonde. Les sondes comprennent des marqueurs fixés de façon non covalente permettant la détection de l'hybridation de la deuxième sonde ou de la troisième sonde avec la séquence d'acide nucléique. L'invention concerne également des nécessaires.
PCT/US2001/016089 2000-05-19 2001-05-17 Detection de mutations et de polymorphismes dans des acides nucleiques WO2001090399A2 (fr)

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002096913A1 (fr) * 2001-05-25 2002-12-05 Gentian As Phtalocyanines de di(hydroxy/alcoxy)silicium substituees et utilisations de ces dernieres
EP1546385A2 (fr) * 2002-09-06 2005-06-29 The Trustees of Boston University Quantification de l'expression genetique
US7105308B2 (en) 2002-07-25 2006-09-12 Monogram Biosciences, Inc. Detecting receptor oligomerization
WO2006097506A2 (fr) * 2005-03-16 2006-09-21 Thomas Koehler Procede et ensemble de test destines a la detection d'acides nucleiques cibles
US7402398B2 (en) 2003-07-17 2008-07-22 Monogram Biosciences, Inc. Measuring receptor homodimerization
US7655399B2 (en) 2003-10-08 2010-02-02 Trustees Of Boston University Methods for prenatal diagnosis of chromosomal abnormalities
US7700325B2 (en) 2003-01-17 2010-04-20 Trustees Of Boston University Haplotype analysis
US7709262B2 (en) 2004-02-18 2010-05-04 Trustees Of Boston University Method for detecting and quantifying rare mutations/polymorphisms
EP2341349A1 (fr) * 2000-05-04 2011-07-06 Siemens Healthcare Diagnostics Products GmbH Methodes servant à la detection d'analytes multiples
CN107022612A (zh) * 2017-04-17 2017-08-08 长沙医学院 一种金黄色葡萄球菌的检测方法

Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1305632B1 (fr) * 2000-05-04 2011-07-27 Siemens Healthcare Diagnostics Products GmbH Methodes servant a la detection d'analytes multiples
EP2341349A1 (fr) * 2000-05-04 2011-07-06 Siemens Healthcare Diagnostics Products GmbH Methodes servant à la detection d'analytes multiples
WO2002096913A1 (fr) * 2001-05-25 2002-12-05 Gentian As Phtalocyanines de di(hydroxy/alcoxy)silicium substituees et utilisations de ces dernieres
US7135300B2 (en) 2002-07-25 2006-11-14 Monogram Biosciences, Inc. Profiling frequencies of receptor heterodimers
US7105308B2 (en) 2002-07-25 2006-09-12 Monogram Biosciences, Inc. Detecting receptor oligomerization
US8034567B2 (en) 2002-09-06 2011-10-11 Trustees Of Boston University Quantification of gene expression
EP1546385A4 (fr) * 2002-09-06 2006-12-20 Univ Boston Quantification de l'expression genetique
US8304194B2 (en) 2002-09-06 2012-11-06 The Trustees Of Boston University Quantification of gene expression
EP1546385A2 (fr) * 2002-09-06 2005-06-29 The Trustees of Boston University Quantification de l'expression genetique
JP2005537804A (ja) * 2002-09-06 2005-12-15 ザ トラスティーズ オブ ボストン ユニバーシティ 遺伝子発現の定量方法
US7700325B2 (en) 2003-01-17 2010-04-20 Trustees Of Boston University Haplotype analysis
US8318460B2 (en) 2003-01-17 2012-11-27 Trustees Of Boston University Haplotype analysis
US8501443B2 (en) 2003-01-17 2013-08-06 Trustees Of Boston University Haplotype analysis
US7402398B2 (en) 2003-07-17 2008-07-22 Monogram Biosciences, Inc. Measuring receptor homodimerization
US7785798B2 (en) 2003-10-08 2010-08-31 Trustees Of Boston University Methods for prenatal diagnosis of chromosomal abnormalities
US7655399B2 (en) 2003-10-08 2010-02-02 Trustees Of Boston University Methods for prenatal diagnosis of chromosomal abnormalities
US7709262B2 (en) 2004-02-18 2010-05-04 Trustees Of Boston University Method for detecting and quantifying rare mutations/polymorphisms
US8153377B2 (en) 2004-02-18 2012-04-10 Trustees Of Boston University Method for detecting and quantifying rare mutations/polymorphisms
US9376714B2 (en) 2004-02-18 2016-06-28 Trustees Of Boston University Method for detecting and quantifying rare mutations/polymorphisms
WO2006097506A2 (fr) * 2005-03-16 2006-09-21 Thomas Koehler Procede et ensemble de test destines a la detection d'acides nucleiques cibles
WO2006097506A3 (fr) * 2005-03-16 2007-05-10 Thomas Koehler Procede et ensemble de test destines a la detection d'acides nucleiques cibles
CN107022612A (zh) * 2017-04-17 2017-08-08 长沙医学院 一种金黄色葡萄球菌的检测方法

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