WO1990003445A1 - Systemes d'amplification/detection d'acides nucleiques cibles - Google Patents

Systemes d'amplification/detection d'acides nucleiques cibles Download PDF

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WO1990003445A1
WO1990003445A1 PCT/US1989/004206 US8904206W WO9003445A1 WO 1990003445 A1 WO1990003445 A1 WO 1990003445A1 US 8904206 W US8904206 W US 8904206W WO 9003445 A1 WO9003445 A1 WO 9003445A1
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nucleic acid
sequence
primer
promoter
target
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PCT/US1989/004206
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English (en)
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Barbara Chen Fei Chu
Gerald Francis Joyce
Leslie Eleazer Orgel
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The Salk Institute For Biological Studies
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Publication of WO1990003445A1 publication Critical patent/WO1990003445A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • 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
    • C12Q1/682—Signal amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6867—Replicase-based amplification, e.g. using Q-beta replicase
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates generally to advances in molecular biology and recombinant DNA technology.
  • the present invention is directed to the methods and means, including assays and pharmaceutical kits containing requisite reagents and means, for detecting in an in vitro or ex vivo setting the presence of nucleic acid species, and by deduction the corresponding polypeptide that nucleic acid encodes, in a biological sample.
  • the present invention features the provision of a molecular switch detection/amplification system that is activated ("switched on”) only in the presence of, by preferential hybridization with, a particular target nucleic acid sequence (i.e., target-activated).
  • nucleic acid sequences characteristic of a particular or general pathogenic disease or condition by the in vitro or ex vivo nucleic acid probe hybridization assays of body fluids and tissues containing requisite target nucleic acid.
  • nucleic acid sequences in a biological sample, in which the said sequences, as so-called target nucleic acid, is present in small amounts relative to its existence amongst a wide variety of other nucleic acid species including RNA, DNA or both.
  • pathological diseases or conditions such as, for example, DNA correlating to that of the human immunodeficiency virus.
  • nucleic acids encoding such viral particles it is desirable to detect other nucleic acids characteristic of a pathological disease or condition such as a defective gene, as in the case of hemophilia, or in the detection of anti-pathogen antibodies of such diseases or conditions.
  • nucleic acids associated with such are present, if at all, in very small amounts relative to total nucleic acid in a given biological sample, such as blood or other body fluid or tissue sample of a given individual to be tested.
  • a reporter system is developed whereby a large number of detectable molecules corresponding to the nucleic acid target are produced for ready detectability and measurement.
  • a reporter system is a signal-generating system associated with the target nucleic acid producing a detectable signal representative of the number of molecules of target sequence.
  • Such systems have employed a chromophore generating moiety linked to a oligonucleotide probe that hybridizes with the target nucleic acid sequence. The chromophore moiety can be isolated from those oligonucleotide probes that have properly hybridized to target, and measured.
  • chromophore generating group is an enzyme such as alkaline phosphatase which has a chro ogenic substrate producing under suitable conditions detectable and measurable colored molecules.
  • Another such system employs radioactive labeling of the nucleic acid probe such that the signal generated by such properly hybridized target nucleic acid can be detected and measured.
  • a second approach has been developed that is fundamentally different in that it involves increasing the copy number of the target nucleic acid sequence itself, in particular in an amount greater than that of nucleic acid sequences with which it is associated in the sample. This can be done by selective amplification of the target nucleic acid sequence.
  • PCR polymerase chain reaction
  • RNAs are known to be susceptible to replication by certain polymerases, such as bacterial phage RNA-dependent RNA polymerase such as Q ⁇ replicase and the replicase from brome mosaic virus (BMV) .
  • the RNA can serve as a sequence template for replication by the RNA polymerase resulting in an amount of replicated RNA sequences that is an exponential increase of the amount initially present.
  • a system in which probe for a target sequence is linked to an RNA capable of being replicated by Q ⁇ replicase is described by Chu et al., Nucleic Acids Research 14. 5591 (1986) and by BMV replicase by March et al., Positive Strand RNA Viruses, Alan R. Liss (Publisher; New York) (1987; Proceedings of UCLA Symposium, 1986) .
  • the first features an oligonucleotide-RNA polymerase adduct, the oligonucleotide portion of which acting as a probe to hybridize with target nucleic acid and the RNA polymerase portion thereof acting as a reporter molecule by using it to initiate transcription of a double stranded DNA having an associated promoter recognized by the RNA polymerase.
  • the resultant replicatable transcripts are then detected and measured.
  • the second features an oligonucleotide- promoter adduct.
  • the oligonucleotide portion again functions as a nucleic acid probe and the promoter functions as a reporter molecule by using it to direct transcription of replicatable RNA encoded DNA having the opposite strand of the promoter operably linked thereto.
  • the third features an oligonucleotide-promoter/DNA molecule adduct.
  • the oligonucleotide portion again functions as a nucleic acid probe and the promoter/DNA molecule as a reporter by associating it with an RNA polymerase.
  • advantage is taken of the natural transcription process (as the first step in .expression of DNA to produce polypeptide products) whereby double-stranded nucleic acid templates containing a promoter sequence recognized by a DNA- dependent RNA polymerase is used to produce a plurality of corresponding RNA transcripts.
  • RNA transcripts can be produced, that are themselves replicatable.
  • particular advantage is taken of the natural primer extension reaction whereby double-stranded nucleic acid templates are used to provide amplification potential based upon use of such templates.
  • the present invention relates to a so-called smart probe as it makes itself detectable only by binding to target. As such, it functions itself as a molecular switch, turning itself on for detection only when necessary, by its binding to its intended target; that is, the adduct hereof is target-activated.
  • the present invention utilizes reporter molecules that are target-activated for use in the detection and measurement of underlying target nucleic acid sequence only when the necessary target nucleic acid sequence is present in the sample tested. It employs only relatively short, hence stable, nucleic acid sequences that need only contain a sequence that can initiate primer extension or transcription and nothing more.
  • the present invention provides a built-in molecular switch means for detecting target nucleic acid sequences that is triggered by the presence of the target nucleic acid sequence itself.
  • the present invention is predicated on the use of an oligonucleotide probe, suitable for hybridization with a segment of a target nucleic acid sequence, that has linked thereto at one end a first nucleic acid moiety that is capable of initiating the production of a primer extension product or a plurality of RNA transcripts, themselves preferably but optionally containing sequence operable for their replication, and at the other end a second nucleic acid moiety sufficiently complementary to said first nucleic acid moiety so as to prevent said first nucleic acid moiety from functioning as a primer or promoter. See Figure 1 hereof for a representative illustration.
  • the present invention thus employs novel adducts of covalently joined moieties, one an oligonucleotide probe capable of hybridizing with a target nucleic acid sequence, and joined at one end thereof a second moiety capable of initiating a primer extension or a transcription process producing a product having the preferred capability of self-replication, and joined at the opposite end of the oligonucleotide probe a third moiety having sufficient reversible hybridization complementarity to said second moiety so as to prevent it from functioning as a primer or promoter.
  • the thus constituted adduct assumes the conformation of a hairpin structure, with the two nucleic acid moieties joined at opposite ends to the oligonucleotide probe being in hybridized association with one another and the oligonucleotide probe moiety being available for hybridization with target nucleic acid sequences.
  • the adduct is contacted with a biological sample that may contain a target nucleic acid sequence that can hybridize with the oligonucleotide probe. If no target sequence is present, the adduct is inert to detection because the moiety capable of initiating a primer extension or transcription event in the process of detection and measurement of the target sequence remains unavailably bound by hybridization to its complement. If a target sequence is present in the sample, it hybridizes to the oligonucleotide forcing apart preferentially the moiety capable of initiating a primer extension or transcription event from its complement. In its freed configuration, the moiety capable of initiating a primer extension or transcription event does so as a target-activated act in the process of detecting and measuring the target sequence. See Figure 1.
  • the present invention is directed to the novel adduct, its preparation and use, having linked moieties:
  • an oligonucleotide probe capable of hybridizing to a target nucleic acid sequence in a sample containing same; (2) linked to one end of said probe a nucleic acid primer or promoter sequence that, when disassociated from its complementary nucleic acid sequence upon hybridization of said probe with target nucleic acid sequence, and when optionally cleaved away from said probe, is capable of initiating primer extension or transcription, and (3) linked to the opposite end of said probe a nucleic acid sequence having sufficient reversible hybridization complementarity with said nucleic acid primer or promoter sequence so as to prevent it from functioning as such.
  • moiety (2) is a primer having been disassociated from its complement upon hybridization of probe (1) with target nucleic acid, it is then capable of initiating a primer extension reaction in the presence of dNTPs, appropriate DNA dependent polymerase and a nucleic acid strand longer than said primer in the primer's 3'- direction and having a complement sequence of said primer.
  • moiety (2) is a promoter having been disassociated from its complement upon hybridization of probe (1) with target nucleic acid, it is then capable of initiating transcription when operably arranged with DNA so as to be recognized by a corresponding RNA polymerase.
  • a preferred embodiment produces a product that is self-amplifiable, such as in the case of transcript products, replicatable transcript products.
  • Such primer extension products or (replicatable) RNA transcripts are then detected and measured in a manner known per se such as via incorporation of, or association with, a chromophore moiety or a radioactively detectable moiety, for example.
  • the present invention is directed to the novel application of the natural principles of (preferential) hybridization of complementary nucleic acid sequences, primer extension, transcript production and in preferred embodiments their replication, for the deduced detection and measurement of corresponding target nucleic acid sequence that may be present in a biological sample containing a mixture of nucleic acids including DNA, RNA or both.
  • the present invention is thus directed to all methods and means associated with the use of a target- activated system for the preparation and use of primer extension products or (replicatable) RNA transcripts that can be amplified and detected as such and measured as a basis for the determination of the amount present, if any, of a corresponding target nucleic acid sequence.
  • the present invention is further directed to associated methods and means for devising assay systems based upon such target-activated adducts and their primer extension or (replicatable) transcript products and to kits incorporating such assay methodology together with the necessary reagents and means for measuring target nucleic acid sequences in a laboratory/clinical setting.
  • the present invention thus reduces to a method useful for the detection of at least one specific nucleic acid target sequence in a sample containing nucleic acid, comprising detecting a primer extension or an optionally replicatable RNA transcript product, said product being the product of primer extension initiated by a primer or a product of transcription initiated by a promoter, said primer or promoter functioning as a reporter molecule associated as an adduct with an oligonucleotide probe capable of hybridizing with said target nucleic acid sequence, said adduct comprising said oligonucleotide probe having linked thereto at one end said primer or promoter and thereto at the opposite end a reversibly hybridizable complement of said primer or promoter and being target-activated upon hybridization of said probe with said target nucleic acid sequence so as to disassociate said primer or promoter from its complement and activate it as a functional primer or promoter molecule.
  • the present invention primarily embodies 1) a target-activated oligonucleotide probe-nucleic acid/complement adduct and 2) imposing a primer extension or transcription step between the production of the activated primer or promoter as a reporter molecule and the, for example replication, event of amplification and 3) using relatively short, stable nucleic acids as such reporter molecules.
  • the replicatability of the replicatable transcripts follows by having disposed within the sequence a sequence that is recognized by replicase enzyme. Other means of amplifying the transcript products are within intellectual reach of the art and/or the literature extant.
  • the present invention further embodies means for measuring the amount of said detected primer extension or (replicatable) transcript products.
  • the present invention is directed to a method useful for the detection of at least one specific nucleic acid target sequence in a sample containing nucleic acid, comprising hybridizing with said target nucleic acid sequence under suitable conditions an oligonucleotide-nucleic acid/complement adduct comprising an oligonucleotide probe corresponding in sequence to a segment of said target sequence and linked thereto at one end a functional length of a strand of primer or promoter sequence and thereto at the opposite end a complement of said primer or promoter sequence, said hybridizing event target-activating said primer or promoter sequence by freeing it from its said complement.
  • oligonucleotide-nucleic acid/complement adduct optionally eliminating excess, non- hybridized (with target nucleic acid sequence) oligonucleotide-nucleic acid/complement adduct, assaying the number of activated primer or promoter sequences associated by hybridization of said probe with said target nucleic acid sequence by using it to direct primer extension or transcription, optionally allowing the primer extension or transcript products to replicate or otherwise become amplified, detecting the primer extension or transcript products.
  • the present invention in application, embodies the detection of said self-replicated or otherwise optionally amplified primer extension or RNA transcript products such as via radio- or chromophore-labeling techniques known per se.
  • the present invention contemplates the detection of target nucleic acid sequence in a sample wherein said target nucleic acid sequence is associated with characteristics of a genetic or pathogenic disease or condition, and particularly those wherein the nucleic acid sequence is a segment of a human virus or is a segment of a defective gene.
  • a genetic or pathogenic disease or condition e.g., those wherein the nucleic acid sequence is a segment of a human virus or is a segment of a defective gene.
  • human diseases that are either the direct result of a genetic defect or are correlated with the presence of a particular genetic allele.
  • the technique described in this application could be used to determine whether or not a given target gene is present in a very small sample of DNA.
  • the technique could also be used to detect mRNA species. It would be useful, for example, in the diagnosis of Cooley's anemia, a disease characterized by the absence of ⁇ globin mRNA.
  • Another potential application is the detection of latent viral infections. DNA from peripheral blood cells could be tested for the presence of HIV-1 (AIDS virus) DNA which has become integrated into the host genome.
  • HIV-1 HIV
  • the present invention contemplates the use of particular promoters such as the bacteriophage T7 promoter and wherein RNA transcripts are produced using T7 RNA polymerase or use of the SP6 promoter and corresponding SP6 RNA polymerase.
  • the present invention is also directed to assay systems and kits embodying same, useful for the detection of at least one specific nucleic acid target sequence in a sample containing nucleic acid, comprising detecting a primer extension or an optionally replicatable transcript product, said product being a product of primer extension initiated by a primer or a product of transcription initiated by a promoter, said primer or promoter functioning as a reporter molecule associated as an adduct with an oligonucleotide probe capable of hybridizing with said target nucleic acid sequence, said adduct comprising said oligonucleotide probe having linked thereto at one end said primer or promoter and thereto at the opposite end a reversibly hybridizable complement of said primer or promoter and being target- activated upon hybridization of said probe with said target nucleic acid sequence so as to disassociate said primer or promoter from its complement and activate it as a functional primer or promoter molecule, and means for hybridizing said probe and utilizing the thereby activated primer or promoter reporter of said
  • FIG. 1 depicts schematically an aspect of this invention: a target nucleic acid sequence (T) , a promoter/DNA molecule (P + MDV1) (depicted as single- stranded) and a novel oligonucleotide-promoter/complement adduct hereof (A) are co-contacted to result in the molecule wherein the target nucleic acid sequence (T) is hybridized (depicted as dotted lines) with the oligonucleotide probe complementary sequence (T') thus breaking the hybridization between P " and P + of A and freeing P " of A to associate by hybridization to P + of P + MDV1.
  • the replicatable transcripts are prepared that can be detected and measured demonstrating the presence of target sequence.
  • DNA probe or primer preparation including DNA synthesis or isolation of sequences from natural source via restriction enzyme cleavage and the tailoring thereof so as to be suitable as such or when linked to other DNA for use as a primer or probe herein; preparation of the linked adducts of oligonucleotides and nucleic acids for use in hybridization as oligonucleotide probe/reporter molecule; hybridization methodology including variations in stringency conditions for producing more or less hybridization certainty depending on the degree of homology of the primer to a target DNA sequence; identification, isolation or preparation of promoters, or more specifically promoters or sites recognized by bacteriophage DNA-dependant RNA polymerase and bacteriophage RNA-dependant RNA polymerase or in the employment of eukaryotic systems, viral DNA- and RNA- dependent RNA polymerases, for example, adenovirus encoded RNA polymerase and brome mosaic virus RNA polymerase; identification, isolation or preparation of RNA polymerases capable of recognizing said promoter
  • promoter is meant a nucleic acid sequence (naturally occurring or synthetically produced or a product of restriction digest) that is specifically recognized by an RNA polymerase that binds to a recognized sequence and initiates the process of transcription whereby an RNA transcript is produced. It may optionally contain nucleotide bases extending beyond the actual recognition site, thought to impart additional stability toward degradation processes, and may also include additional plus (+) nucleotides contiguous to the transcription initiation site.
  • any promoter sequence may be employed for which there is a known and available polymerase that is capable of recognizing the initiation sequence. Typical, known and useful promoters are those that are recognized by certain bacteriophage polymerase such as bacteriophage T3, T7 or SP6. See Siebenlist et al. , Cell 20. 269 (1980) . These are but examples of those polymerases that can be employed in the practice of the present invention in conjunction with their associated promoter sequences.
  • the promoter herein is the reporter molecule, it is defined because it exists, as a single- stranded version of an otherwise fully operable, classically defined, double-stranded promoter as given immediately above.
  • RNA transcript hereof is the ribonucleic acid sequence produced after transcription initiation following RNA polymerase recognition of the promoter sequence (See supra) .
  • the production of such transcripts is more or less continuous, dependent in part on the amount of polymerase present.
  • probe in the present context is meant a nucleic acid sequence (naturally occurring or synthetically produced or a product of restriction digest) that has sufficient homology with the target sequence such that under suitable hybridization conditions it is capable of hybridizing, that is binding to, the target sequence.
  • a typical probe is at least about 10 nucleotides in length, and most preferably is of approximately 35 or more nucleotide bases in length, and in its most preferred embodiments, it shares identity or very high homology with the target sequence. See, for example, EPA 128042 (publd. 12 Dec 84) .
  • reversible hybridization complementarity specifically refers to the complementarity between the primer or promoter moiety of adduct and its complement such that they hybridize into a hairpin structure (See A of Figure 1 where P * and P + are hybridized (dotted lines) ) or are separated (hence reversible hybridization) upon hybridization of the oligonucleotide probe with its target nucleic acid complement sequence (again see Figure 1 for the preferential hybridization of T with T' of A to separate P " and P + of A) . The hybridization is sufficient to prevent the primer or promoter from functioning as such.
  • primer herein is meant a sequence of nucleic acid that is at least about 10 nucleotide bases or of otherwise sufficient length such that it will hybridize to a complement sequence for purposes of a primer extension reaction in the presence of appropriate reagents and conditions.
  • the term "operably linked" in particular in connection with the linkage of a promoter sequence within an RNA encoding DNA sequence refers to its functionality in producing corresponding RNA transcripts when the promoter is recognized by the suitable polymerase—see supra.
  • the novel adduct hereof may contain additional bases or sequences that do not interfere with its use but which may lend additional stability to the adduct and/or may work to suppress unwanted side reactions.
  • additional bases may be added to the complement of the sequence capable of initiating transcription that will not hybridize with the sequence capable of initiating transcription and will not interfere with the hybridization of said sequence with its complement but will prevent the complement from itself functioning as a promoter. These would extend from the 5'-end of P + as depicted by "Y" in Figure 1, for example.
  • the principle aspect of the present invention is satisfied by the hybridization of the two complementary ends of the adduct which is preferentially disrupted upon hybridization of the oligonucleotide probe moiety of the adduct with target nucleic acid sequence.
  • This event target-activates the adduct for detection proof.
  • the activation of the sequence capable of initiating primer extension or transcription (P " in Figure 1, for example) is an endpoint of this invention, and it will therefore be understood that in the case of a promoter, the freed, activated sequence can be associated with its opposite strand to form a duplex, making it operable in initiating transcription upon contact of the duplex with an appropriate RNA polymerase.
  • the opposite strand can be associated with virtually any sequence from which products can be obtained that are susceptible to some sort of detection and/or measurement capability, and preferably amplification potential.
  • One such sequence is illustrated herein as a model example, namely, the MDV1 sequence that encodes replicatable RNA.
  • the resultant primer extension products can be chosen such that they are susceptible to amplification and detection/measurement capability in a variety of ways.
  • the primer extension products being double-stranded DNA duplexes, could be subjected to amplification using the so-called PCR method that is known per se—see reference Supr .
  • a sample on which the assay method of the invention is carried out can be a raw specimen of biological material, such as serum or other body fluid, tissue culture medium or food material. More typically, the method is carried out on a sample which is a processed specimen, derived from a raw specimen by various treatments to remove materials that would interfere with detection of target, such as by causing non-specific binding of affinity molecules. Methods of processing raw samples to obtain a sample more suitable for the assay methods of the invention are well known in the art.
  • the method can be carried out on nucleic acid from cells following the colony hybridization method of Grunstein et al. Proc. Natl. Acad. Sci. (U.S.A. ) 72. 3961 (1975) (see also, U.S. Patent Nos. 4,358,535 and 4,562,159) or the plaque lift method of Benton et al.. Science 196. 180 (1977) . It can also be carried out on nucleic acids isolated from viroids, viruses or cells of a specimen and deposited onto solid supports
  • the method can also be carried out with nucleic acid isolated from specimens and deposited on solid support by "dot” blotting (Kafatos et al. , Nucl. Acids Res. 1_, 1541 (1979); White et al. , J. Biol. Chem. 257, 8569 (1982) ; Southern blotting (Southern. J. Mol. Biol. 98. 503 (1975) ; "northern” blotting (Thomas, Proc. Natl. Acad. Sci. (U.S.A.) 77.
  • Nucleic acid of specimens can also be assayed by the method of the present invention applied to water phase hybridization (Britten et al. , Science 161. 527 (1968)) and water/organic interphase hybridizations (Kohne et al.. Biochemistry 16, 5329
  • Water/organic interphase hybridizations have the advantage of proceeding with very rapid kinetics but are not suitable when an organic phase-soluble linking moiety, such as biotin, is joined to the nucleic acid affinity molecule.
  • the assay method of the invention can also be carried out on proteins or polysaccharides isolated from specimens and deposited onto solid supports by dot-blotting, by "Western” blotting or by adsorption onto walls of microliter plate wells or solid support materials on dipsticks. Still further, the method of the invention is applicable to detecting cellular proteins or polysaccharides on the surfaces of whole cells from a specimen or proteins or polysaccharides from microorganisms immobilized on a solid support, such as replica-plated bacteria or yeast.
  • bacteriophage Q ⁇ is not limited to any particular variant or mutant or population thereof. Such reference, unless otherwise specifically limited, is to any variant, mutant or population which, upon infection therewith of E. coli susceptible to bacteriophage Q ⁇ infection, is capable of causing production of an RNA-dependent RNA-polymerase.
  • RNA-dependent RNA polymerases for other phages which, upon infection of bacteria susceptible to infection therewith, produce RNA-dependent RNA polymerases, and associated replicatable RNAs capable of being autocatalytically replicated in vitro, which can be employed in the present invention, see, e.g., Miyake et al.. Proc. Natl. Acad. Sci. (U.S.A.) 68. 2022 (1971) .
  • covalent linkages include, among others, the following:
  • Linking moiety is a phosphate group and linkage is directly between the phosphate and the 5'-carbon of the 5'-nucleotide of replicative RNA.
  • the phosphate linking moiety, bonded to the 5'-carbon of the 5'-nucleotide of replicative RNA, will usually be involved in covalently joining a replicative RNA directly to the 3'-carbon of the 3'-nucleotide of a nucleic acid affinity molecule or to the 3'-carbon of the 3'-nucleotide of a segment of nucleotides which is a linking moiety considered to be bonded to the 3'-end of a nucleic acid affinity molecule and which is covalently joined, through a phosphite at the 5'-carbon of its 5'-nucleotide, to the 3'-carbon of the 3'-nucleotide of the affinity molecule.
  • the 5'-terminal nucleotide of a replicative RNA can be phosphorylated at the 5'-carbon with T4 polynucleotide kinase by methods known in the art.
  • Affinity molecule, or nucleic acid linking moiety of affinity molecule can then be connected to the 5'-phosphate of the 5'-nucleotide of replicative RNA by known methods employing T4 RNA ligase.
  • Linking moiety is biotinyl or iminobiotinyl .and linkage is to the 5'-carbon of the
  • Replicative RNA with spacer group of formula -NH(C0 2 ) aa NH(P0 2 )0- can be made following the teaching of Chu and Orgel, DNA 4 . , 327 (1985) .
  • Replicative RNA with spacer group of formula -NH(CH 2 ) bb SS(CH 2 ) cc NH(P0 2 )0- is taught in Example I.
  • Replicative RNA with spacer group of formula -NH(CH 2 ) bb (CO) (NH) (CH 2 ) cc NH(P0 2 )0- is made by reacting replicative RNA, with group of formula
  • RNA can be detected in a number of different ways: Detection can be by ultraviolet absorbance of replicated RNA, as, for example, by the method of contact photoprinting (Kutateladze et al.. Anal. Biochem. loo, 129 (1979)).
  • the replicated RNA can be detected, by any of numerous known procedures, by means of its radioactivity.
  • Biotin or iminobiotin can be incorporated into replicated RNA, which can then be detected by known techniques with an enzyme-avidin or enzyme-streptavidin adduct, which binds to the RNA-bound biotin and catalyzes production of a conveniently detectable chromogen.
  • UTP that is biotinylated through a spacer to carbon-5 of the uracil moiety as a substrate for the replicase in the replication reaction.
  • UTP's are known compounds. Further, it is known that such UTP's are substrates for Q ⁇ replicase, and that RNAs which include uracils biotinylated through spacer groups joined to the carbon-5 position, due to use of such UTP's in their synthesis, are templates for Q ⁇ replicase catalyzed replication.
  • RNA resulting from the replication process could also be biotinylated employing photobiotin acetate and then detected, with an avidin-enzyme adduct-chromogenic compound system, like replicated RNA's synthesized with biotinylated UTP in the replication reaction.
  • RNA resulting from the replication process can be made fluorescent by employing a T4 RNA ligase catalyzed reaction to append nucleotides modified to be fluorescent to the 3'-end of replicative RNA. See Cosstick et al.. Nucl. Acids Res. 12. 1791 (1984) .
  • the fluorescence of the resulting RNA can be employed to detect the RNA by any of several standard techniques.
  • RNA RNA binds specifically with nucleic acid
  • a reporter substance that binds specifically with nucleic acid
  • the medium such as a positively charged support such as ECTE0LA paper
  • ECTE0LA paper a positively charged support
  • Such substances include: chromogenic dyes, such as "stains all" (Dahlberg et al. f J. Mol. Biol. 41. 139 (1969) ; methylene blue (Dingman et al.. Biochemistry , 659 (1968) , and silver stain (Sammons et al.
  • RNAs that are templates for replication by Q ⁇ replicase for example, a phycobiliprotein (Oi et al.. J. Cell Biol. 93. 981 (1982); Stryer et al. , U.S. Patent No.
  • the concentration of template RNA, in a replicase-catalyzed replication reaction system after a given time for reaction, will be related to the initial concentration of template RNA. If, at all times during the replication reaction, the concentration of replicase exceeds that of template (and ribonucleoside-5'- triphosphate concentration does not become limiting) , the log of concentration of template RNA at the conclusion of the reaction will be directly proportional to the log of the initial concentration of template (at the start of the reaction) . After replicase concentration falls below template concentration, as long as ribonucleoside-5'-triphosphate concentration does not become limiting, the concentration of template at the conclusion of reaction is directly proportional to the log of the initial concentration of template. Further, the time required for a reaction to reach the point at which template concentration equals replicase concentration is proportional to the negative log of the initial concentration of template.
  • assays are carried out simultaneously, under conditions as nearly alike as possible, on both test samples, which are being tested for target, and control samples.
  • control samples are similar to test samples but are known to contain either no target or a known quantity of target.
  • a control with no target establishes the "background,” below which it is not possible to distinguish samples which contain target from those which do not.
  • replicases for (autocatalytic) induction of replication of the RNA transcripts of the present invention are generally known in the art.
  • Suitable examples of such replicases that are useful in the present invention include the so-called Q ⁇ virus replicase that recognizes certain nucleic acid sequence sites at both the 3'- and 5'- ends of the given RNA transcript and the so-called brome mosaic virus (BMV) as well as the alpha virus replicases which are thought to recognize nucleic acid sequence sites at the 3' " end of a given RNA transcript.
  • BMV brome mosaic virus
  • These replicases serve to replicate, that is reproduce, the RNA transcripts and complements so as to multiply copies thereof.
  • the multiple transcripts that are produced during transcription can themselves undergo replication so as to exponentially increase the amount of RNA transcript product.
  • the target nucleic acid in a sample is probed using an oligodeoxynucleotide adduct that contains three subsequences: (1) a complement sequence of the target sequence (T' in Figure 1) , (2) the appropriate single strand (minus strand) of the promoter for T7 RNA polymerase (P " in Figure 1) , and (3) a complement sequence of (2) (P + in Figure 1) .
  • the assay system may contain adduct (A) , the biological sample suspected of harboring the target nucleic acid sequence and the DNA encoding RNA, for example P + MDV1. When the adduct is subjected to hybridization conditions alone, it folds into the hairpin structure shown by A in Figure 1 or forms an intermolecular complex.
  • probe-promoter/complement adducts hereof are “smart probes,” that is, the promoter is activated for use if and only if the probe portion is hybridized to its target. In effect, the probe is “smart” because it makes itself detectable by binding to target.
  • the adduct consists essentially of three segments: a probe segment, which has a sequence complementary to that of the segment of target to which the probe hybridizes, a "5'-clamp” segment, which extends from the 5'-nucleotide of the probe segment and encodes one (+) strand of a promoter, and a "3'-clamp” segment which extends from the 3'-nucleotide of the probe segment and is a complement of the 5'-clamp and encodes the (-) strand of a promoter.
  • the promoter is "clamped" in a non-active form, inactive to initiate transcription as long as the 3'-clamp segment and the 5'-clamp segment are hybridized.
  • the clamp segments release and the adduct snaps into an activated form. It is not necessary to displace adduct that has not hybridized to target sequence by washing as it is inert to utility with the P + MDV1, for example.
  • Hybridized material is released from the target nucleic acid by simple denaturing and/or by displacement using an oligodioxinucleotide with greater affinity for the target.
  • the portion that is complementary to the T7 promoter can be released by chemical methods, leaving the remaining portion bound to the target.
  • a cleavable linker for example, a disulfide bond
  • the released DNA that contains the complement of the T7 promoter serves as a reporter molecule for successful hybridization events.
  • This DNA being hybridized to a single-stranded DNA molecule that contains the (plus) strand of the T7 promoter joined to a sequence that codes for an RNA substrate of Q ⁇ RNA polymerase, is a functional double-stranded T7 promoter joined to a single- or double-stranded template encoding an RNA transcript.
  • the T7 RNA polymerase binds to the double-stranded promoter and proceeds to transcribe the template (see Milligan et al., Nucleic Acids Research 15, 8783 (1987)).
  • the resulting RNA is assayed using Q ⁇ RNA polymerase exactly as is described in the patent application cited supra.
  • T7 RNA polymerase is a DNA-dependent RNA polymerase that has the following useful properties:
  • the enzyme can operate on either single- or double- stranded templates;
  • the enzyme has a high turnover rate, producing 200-1200 moles of RNA transcript per mole of DNA template
  • MDV-1 (+) RNA contains 221 nucleotides, beginning with the sequence GGG at its 5' end. It in turn serves as an ideal substrate for Q ⁇ RNA polymerase, an RNA-dependent RNA polymerase which carries out autocataly.tic amplification of its substrate RNA. Combining the T7 RNA polymerase system with the Q ⁇ RNA polymerase system provides an extremely powerful tool for amplifying the signal generated by a rare molecular event.
  • T The target sequence 5'-GTTGTGTGGAATTGTG-3' (T) which is part of the sequence of the M13mp8 (+) strand DNA is detected.
  • T' the complement of T, is linked to the minus strand of the promoter at one end and the complement of the promoter at its opposite end and uses the general principle of the invention to detect T by detecting the production of MDV-1 RNA produced in a suitable assay.
  • the linkage of probe to the minus strand of the promoter and to its complement may be accomplished in two ways: 1) by normal phosphodiester linkage.
  • the following sequence will be synthesized in a DNA synthesizer:
  • the 5 7 -phosphate derivatives (0.1-10 ODUJare converted to the 5'-cystamine derivatives by treatment with 0.1 M 1-methylimidazole, 0.15 M l-ethyl-3 ,3-dimethylaminopropyl carbodiimide and 0.5 M cysta ine at pH 7 and 50°C for 2 hours.
  • the 5'- cystamine derivatives are purified either by HPLC on RPC- 5 or denaturing gel electrophoresis.
  • a mixture containing the 5'-cystamine derivatives of sequences 1, 2 and 3 is treated with DTT in TRIS-EDTA buffer at pH 7.2 for 1 hour at room temperature.
  • the reaction mixture is then dialyzed against buffer containing 0.1 mM DTT, ImM Tris and 0.1 mM EDTA at pH 7.2 for 30 ins, and against fresh buffer containing 1 mM Tris and 0.1 mM EDTA at pH 7.2 for a further 30 mins.
  • the mixture is then concentrated, if necessary in a speed-vac concentrator and the probe- promoter/complement adduct purified by gel electrophoresis.
  • the nitrocellulose blots are pre-hybridized for 1 hour at 30°C in hybridization buffer (900 mM NaCl, 6 mM EDTA, 90 mM Tris pH 7.5, 0.1% SDS) containing 100 ⁇ g/ml randomly cleaved RNA.
  • Hybridization with 1 ng/ml of the probe-promoter/complement adduct is then carried out at 45°C for 1 hour.
  • the blots are then washed twice with buffer containing 180 mM NaCl at room temperature and again with buffer containing 18 mM NaCl at 30°C.
  • the probe-promoter/complement linked by phosphodiester bonds will be released from the target slots in 30 ⁇ l boiling buffer, cooled at room temperature for 15 mins.
  • the probe linked to the promoter/complement by disulfide bonds will be released by incubation of the target slot with 30 ⁇ l of 10 mM DDT in Tris-EDTA buffer at 37 " C for 1 hour.
  • Hybridization of the Released Promoter The released DNA containing the minus strand of the T7 promoter, serving as a reporter for successful target hybridization events, is hybridized to a single- stranded DNA molecule which contains the 17-nucleotide plus (+) strand of the T7 promoter joined to a 221- nucleotide sequence which codes for MDV-1 RNA. Hybridization occurs in a 40 ⁇ l volume which contains 1 pmole (-100 ng) T7 promoter-MDV-1 DNA, 12 mM MgCl 2 , 2mM spermidine, and 50 mM Tris (pH 7.5) . This mixture is heated to 65-C for 5 min and then cooled to 30°C over 5- 10 min.
  • RNA is determined by its intrinsic UV absorbance (e.g. as by the contact photoprinting method of Kutateladze et al. , Anal. Biochem. 100. 129 (1979)).
  • RNA is visualized on ETEOLA paper.
  • Aliquots (of equal volume) of replication reaction are transferred with 13, 48 or 96-fingered aliquotter to sheets of diethylaminoethyl cellulose paper.
  • the sheets are then washed at room temperature in a solution of 200 mM NaCl, 300 mM ammonium acetate pH 6 to remove ribonucleosides not incorporated into RNA.
  • the sheets are then stained with 0.3 ⁇ g/ml of ethidium bromide. (Sharp et al. , Biochemistry 12. 3055 (1973); Bailey et al. , Anal. Biochem 70. 75 (1976) .
  • fluorescence intensity from a stained blot above that from control blots indicates the presence of target.
  • Other staining materials can be employed in place of ethidium bromide. These include methylene blue (Dingman and Peacock, Biochemistry , 659 (1968)), silver stain (Sammons, et al.. Electrophoresis 2. 135 (1981)) or phycobiliprotein Q ⁇ replicase conjugate (Oi et al.. J. Cell Biol. 93. 981 (1982)).
  • Rubella antibody is detected in a patient with recent exposure to rubella antigen.
  • Microliter wells coated with rubella antigen are incubated for 3 hours at room temperature with 50 ⁇ l aliquots per well of 1:10, 1:30, 1:100, 1:300, 1:1000, and 1:3000 dilutions of IgG isolated from the patient. Dilutions are prepared with 5% horse serum in phosphate-buffered saline. The plates are then thoroughly washed with Tween 20-NaCl. To each well is then added 50 ⁇ l of a solution containing 1 ⁇ g/ml of anti-rubella IgG linked by disulfide bonds to the minus strand of the promoter and its complement.
  • Anti- rubella IgG is first thiolated with imino-thiolane and then reacted with the 5'-(2-pyr)-SS-P-sequences 2 and 3 to give the disulfide linked adduct: 5'-promoter + -3'-P-CH 2 CH 2 -SS-
  • Unreacted IgG is eluted with 50 M Tris at pH 7, and the IgG-promoter/ complement -adduct is eluted with the same buffer containing 0.25 M NaCl. Unreacted oligonucleotide can be eluted with buffer containing 0.5 M NaCl.

Abstract

Cette invention concerne l'utilisation de molécules rapporteuses fonctionnelles lors de la détection et la mesure des séquences d'acides nucléiques dans un échantillon pour déterminer, par exemple, l'existence ou le potentiel de maladies pathogéniques. L'invention concerne l'utilisation d'un produit d'addition ayant une séquence de sonde d'oligonucléotides liée à (1) une séquence pouvant initier une extension ou une transcription d'amorce et (2) un complément de (1). Le produit d'addition possède une structure d'épingle à cheveux rendant la séquence de (1) inactive. Lors de l'hybridation de la séquence de sonde d'oligonucléotide avec une séquence d'acide nucléique cible, le produit d'addition est activé par la cible et la séquence (1) est disponible pour détecter et mesurer la séquence d'acide nucléique cible.
PCT/US1989/004206 1988-09-30 1989-09-28 Systemes d'amplification/detection d'acides nucleiques cibles WO1990003445A1 (fr)

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EP0427073A2 (fr) * 1989-11-09 1991-05-15 Miles Inc. Amplification d'acide nucléique employant une sonde liante en forme d'épingle à cheveux et transcription
WO1992018522A1 (fr) * 1991-04-18 1992-10-29 The Salk Institute For Biological Studies Oligodesoxynucleotides et oligonucleotides utiles en tant que pieges pour des proteines qui se lient selectivement a des sequences definies d'adn
EP0601889A2 (fr) * 1992-12-10 1994-06-15 Maine Medical Center Research Institute Sondes d'acides nucléiques
US5985548A (en) * 1993-02-04 1999-11-16 E. I. Du Pont De Nemours And Company Amplification of assay reporters by nucleic acid replication
US6485903B1 (en) 1995-05-05 2002-11-26 Pe Corporation (Ny) Methods and reagents for combined PCR amplification and hybridization probing
US7833716B2 (en) 2006-06-06 2010-11-16 Gen-Probe Incorporated Tagged oligonucleotides and their use in nucleic acid amplification methods

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US4710464A (en) * 1984-09-27 1987-12-01 Eli Lilly And Company Transcription terminators
US4725536A (en) * 1985-09-19 1988-02-16 Genetics Institute, Inc. Reagent polynucleotide complex with multiple target binding regions, and kit and methods
EP0224126A2 (fr) * 1985-11-25 1987-06-03 The University of Calgary Oligodéoxynucléotides complémentaires liées par covalence utilisées comme "primer linkers" universels pour la détermination de la séquence d'acides nucléiques

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NUCLEIC ACIDS RESEARCH, Volume 14, Number 17, published in 1986, (IRL Press Limited, Oxford, England); S. KRAWETZ et al.: "Covalenty linked complementary oligodeoxynucleotides (splinkers) as tools for molecular biology", page 7131, see especially page 7131, lines 1-28. *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES (USA), Volume 85, published in 1988, (The National Academy of Sciences, Washington D.C); D.ENGELKE et al.: "Direct sequencing of enzymatically amplified human genomic DNA", pages 544-548, see especially the Abstract on page 544. *

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0427073A2 (fr) * 1989-11-09 1991-05-15 Miles Inc. Amplification d'acide nucléique employant une sonde liante en forme d'épingle à cheveux et transcription
EP0427073A3 (en) * 1989-11-09 1991-08-28 Molecular Diagnostics, Inc. Nucleic acid amplification employing ligatable hairpin probe and transcription
WO1992018522A1 (fr) * 1991-04-18 1992-10-29 The Salk Institute For Biological Studies Oligodesoxynucleotides et oligonucleotides utiles en tant que pieges pour des proteines qui se lient selectivement a des sequences definies d'adn
US5683985A (en) * 1991-04-18 1997-11-04 The Salk Institute For Biological Studies Oligonucleotide decoys and methods relating thereto
EP0601889A2 (fr) * 1992-12-10 1994-06-15 Maine Medical Center Research Institute Sondes d'acides nucléiques
EP0601889A3 (fr) * 1992-12-10 1994-08-03 Maine Medical Center Res
US5607834A (en) * 1992-12-10 1997-03-04 Maine Medical Center Research Institute Fluorescent imperfect hairpin nucleic acid probes
US5985548A (en) * 1993-02-04 1999-11-16 E. I. Du Pont De Nemours And Company Amplification of assay reporters by nucleic acid replication
US6485903B1 (en) 1995-05-05 2002-11-26 Pe Corporation (Ny) Methods and reagents for combined PCR amplification and hybridization probing
US7241596B2 (en) 1995-05-05 2007-07-10 Applera Corporation Methods and reagents for combined PCR amplification and hybridization probing
US7413708B2 (en) 1995-05-05 2008-08-19 Applied Biosystems Inc. Methods and reagents for combined PCR amplification
US7847076B2 (en) 1995-05-05 2010-12-07 Life Technologies Corporation Methods and reagents for combined PCR amplification
US7833716B2 (en) 2006-06-06 2010-11-16 Gen-Probe Incorporated Tagged oligonucleotides and their use in nucleic acid amplification methods
US8034570B2 (en) 2006-06-06 2011-10-11 Gen-Probe Incorporated Tagged oligonucleotides and their use in nucleic acid amplification methods
US8278052B2 (en) 2006-06-06 2012-10-02 Gen-Probe Incorporated Tagged oligonucleotides and their use in nucleic acid amplification methods
US8580510B2 (en) 2006-06-06 2013-11-12 Gen-Probe Incorporated Tagged oligonucleotides and their use in nucleic acid amplification methods
US9284549B2 (en) 2006-06-06 2016-03-15 Gen-Probe Incorporated Tagged oligonucleotides and their use in nucleic acid amplification methods
US10167500B2 (en) 2006-06-06 2019-01-01 Gen-Probe Incorporated Tagged oligonucleotides and their use in nucleic acid amplification methods
USRE48909E1 (en) 2006-06-06 2022-02-01 Gen-Probe Incorporated Tagged oligonucleotides and their use in nucleic acid amplification methods

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