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WO2000070095A2 - Amplification isothermique homogene et detection d'acides nucleiques utilisant un oligonucleotide de commutation de matrice - Google Patents

Amplification isothermique homogene et detection d'acides nucleiques utilisant un oligonucleotide de commutation de matrice Download PDF

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Publication number
WO2000070095A2
WO2000070095A2 PCT/US2000/013526 US0013526W WO0070095A2 WO 2000070095 A2 WO2000070095 A2 WO 2000070095A2 US 0013526 W US0013526 W US 0013526W WO 0070095 A2 WO0070095 A2 WO 0070095A2
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sequence
target
primer
dna
nucleic acid
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WO2000070095A3 (fr
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Nurith Kurn
Yen Ping Liu
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Dade Behring Inc
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Dade Behring Inc
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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
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • 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
    • 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
    • C12Q1/6865—Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]

Definitions

  • the present invention relates to the detection of differences in nucleic acids using a method for isothermal amplification of polynucleotide sequences.
  • the amplification method uses template switch oligonucleotide which subsequently allows for the detection of the presence of a difference between a target polynucleotide sequence and a reference polynucleotide sequence.
  • Nucleic acid hybridization has been employed for investigating the identity and establishing the presence of nucleic acids. Hybridization is based on complementary base pairing. When complementary single stranded nucleic acids are incubated together, the complementary base sequences pair to form double stranded hybrid molecules. The ability of single stranded deoxyribonucleic acid (ssDNA) or ribonucleic acid (RNA) to form a hydrogen bonded structure with a complementary nucleic acid sequence has been employed as an analytical tool in molecular biology research.
  • ssDNA single stranded deoxyribonucleic acid
  • RNA ribonucleic acid
  • Nucleic acid hybridization has great potential in diagnosing disease states associated with unique nucleic acid sequences. These unique nucleic acid sequences may result from genetic or environmental change in DNA by insertions, deletions, point mutations, or by acquiring foreign DNA or RNA by means of infection by bacteria, molds, fungi, and viruses. Nucleic acid hybridization has, until now, been employed primarily in academic and industrial molecular biology laboratories. The application of nucleic acid hybridization as a diagnostic tool in clinical medicine is limited because of the frequently very low concentrations of disease related DNA or RNA present in a patient's body fluid and the unavailability of a sufficiently sensitive method of nucleic acid hybridization analysis
  • One method for detecting specific nucleic acid sequences generally involves immobilization of the target nucleic acid on a solid support such as nitrocellulose paper, cellulose paper, diazotized paper, or a nylon membrane After the target nucleic acid is fixed on the support, the support is contacted with a suitably labeled probe nucleic acid for about two to forty-eight hours After the above time period, the solid support is washed several times at a controlled temperature to remove unhyb ⁇ dized probe The support is then dried and the hybridized material is detected by autoradiography or by spectromet ⁇ c methods When very low concentrations must be detected, this method is slow and labor intensive, and nonisotopic labels that are less readily detected than radiolabels are frequently not suitable
  • RNA target is initiated by hybridization of a primer which is 5'-ta ⁇ led by a sequence representing one strand of a DNA-dependent RNA polymerase promoter
  • the hybridization complex serves as a priming complex for a reverse transc ⁇ ptase to produce an RNA-DNA heteroduplex
  • the RNA strand of the heteroduplex is degraded by RNase H to produce a single-stranded DNA product
  • a second reverse primer hybridizes to the newly formed DNA molecule at a site downstream to the first primer sequence and is extended to form a double-stranded DNA molecule
  • This process produces a double-stranded promoter site for the DNA-dependent RNA polymerase which in turn produces single-stranded RNA products, which are anti-sense to the initial target
  • the rate of initiation of this process is not well-controlled and could pose a problem when attempting to quantify a target nucleic
  • RNA viruses in the presence of host cells which are likely to contain integrated viral genes are likely to contain integrated viral genes
  • nucleic acid probes A method utilizing such probes is described in U S Patent No 4,868,104
  • a nucleic acid probe may be, or may be capable of being, labeled with a reporter group or may be, or may be capable of becoming, bound to a support Detection of signal depends upon the nature of the label or reporter group If the label or reporter group is an enzyme, additional members of the signal producing system include enzyme substrates and so forth
  • PCT application WO 97/23646 describes a method for detection of sequence alteration based on inhibition of DNA branch migration This method is based on inhibition of spontaneous strand exchange by branch migration in four-stranded DNA cruciform structures when a test sequence is altered relative to a reference sequence
  • the substrates are produced by PCR amplification of test and reference DNA sequences using specifically modified primers Any sequence alterations, such as base substitutions, deletions, and insertions are equally detected, and the method is useful for the detection of sequence alterations in heterozygote and homozygote genotypes In addition to its potential usefulness for the diagnosis of genetic disease the method is also useful for the determination of sequence identity required for various applications
  • the branch migration inhibition method for detection of sequence alteration requires the formation of amplification products which are capable, upon denaturation and re-association of forming partial duplexes which in turn anneal to form four- stranded cruciform structures
  • amplification products capable, upon denaturation and re-association of forming partial duplexes which in turn anneal to form four- stranded cruciform structures
  • strand exchange by spontaneous branch migration proceeds if the test and reference amplification products are identical
  • branch migration is inhibited, resulting in the formation of stable, detectable four stranded cruciform structures
  • Ligase Chain Reaction is described in European Patent Application No 0320308B1 , as well as Wu D et al, The Ligation Amplification Reaction (LAR) - Amplification of Specific DNA Sequences Using Sequential Rounds of Template- Dependent Ligation, Genomics, A 560-569 (1989) and Barany, F , Genetic disease detection and DNA amplification using cloned thermostable ligase, Proc Natl Acad
  • Nucleic acid amplification using single polynucleotide primer is described in U S Patent No 5,595,891
  • a method for producing a single stranded polynucleotide having two different defined sequences and kits for use in ASPP is described in U S Patent No 5,683,879 and U S Patent No 5,679,512
  • TMA Transcription based amplification of nucleic acid sequences is described in U S Patent No 5,766,849 (TMA) and U S Patent No 5,654,142 (NASBA)
  • Electrochemiluminescence-based detection is described by DiCesare, J et al, A High-Sensitivity Electrochemiluminescence-Based Detection System for Automated PCR Product Quantitation, BioTechniques, 15(1) 152-157 (1993)
  • PCR-based assay that utilizes the inherent 5' nuclease of rTth DNA polymerase for the quantitative detection of HCV RNA is disclosed by Tsang, et al , (94th General Meeting of the American Society for Microbiology, Las Vegas NE 5/94,
  • German patent application DE 4234086-A1 (92 02 05) (Henco, et al ) discusses the determination of nucleic acid sequences amplified in vitro in enclosed reaction zone where probe(s) capable of interacting with target sequence is present during or after amplification and spectroscopically measurable parameters of probe undergo change thereby generating signal
  • Padlock probes circularizing oligonucleotides for localized DNA detection, are described by Nilsson, et al Science 265 2085-2088 (1994) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase is described by Saiki, et al , Science, 239 487 (1988)
  • U S Patent No 5,508,178 describes nucleic acid amplification using a single polynucleotide primer (ASPP)
  • U S Patent No 5,595,891 discloses methods for producing a polynucleotide for use in single primer amplification
  • U S Patent No 5,439,793 describes a method for producing a molecule containing an intramolecular base-pair structure
  • a method for producing a polynucleotide for use in single primer amplification is described in U S Patent No 5 612,199
  • a method for introducing defined sequences at the 3'-end of a polynucleotide is described in U S Patent No
  • the present invention provides for a method of producing multiple copies of a nucleic acid sequence involving the step of combining in a target polynucleotide, a first ohgonucleotide primer a template switch ohgonucleotide, and reagents sufficient for conducting and amplification of the polynucleotide sequence
  • the combination is subjected to conditions for amplifying the polynucleotide sequence
  • the template switch ohgonucleotide includes a 3' region capable of hybridizing to the target and a 5' region which does not hybridize to the target
  • the 5' region includes (1) a propromoter sequence of a DNA dependant RNA polymerase, (2) a sequence substantially homologous to the target sequence located 3' of the propromoter sequence, and (3) a region unrelated to the target sequence located between the propomoter sequence and the sequence homologous to the target
  • the invention further provides for the addition of a second primer unrelated to the target sequence
  • the second primer includes a sequence substantially homologous to the 5 sequence of the template switch ohgonucleotide which is unrelated to the target
  • the invention provides for a denaturation step
  • a first primer and a template switch ohgonucleotide are hybridized to the same strand target
  • the first primer is extended along the target and then along the template switch ohgonucleotide to form a complex comprising a first extension product and the promoter for a DNA dependent RNA polymerase
  • the first extension product is transcribed to produce multiple copies of a first transcription product which includes a sequence substantially homologous to the target
  • the first primer may hybridize to the transcription product and be extended to form an RNA/DNA heteroduplex comprising a second extension product After degradation of the transcription product the second primer is hybridized to the second extension product and is extended to form a third extension product The second extension product is also extended to produce a fully double stranded DNA product with the third extension product The double stranded DNA product is transcribed to produce multiple copies of the transcription product which includes a sequence substantially homologous to the target
  • Another aspect of the invention involves the detection of the presence of a difference between a target nucleic acid sequence and a reference sequence
  • the target and the reference nucleic acid sequences are amplified using a template switch ohgonucleotide, a first primer, a second primer and a third primer
  • the first and second primers have common 3' sequences which are complementary to the target sequence and the reference sequence, and 5' tails which are not complementary to the target, the reference, or each other
  • the first primer has a first label and the second primer has a second label
  • the third primer is a mixture of the third primer with the first label and the third primer with the second label
  • a complex is formed including the reference sequence and the target sequence in double stranded form
  • the complex has at least one pair of non- complementary strands and each of the non-complementary strands has a label
  • the complex is subjected to strand exchange conditions wherein if no difference exists between the reference sequence and the target sequence, strand exchange continues until complete If
  • Labels useful for the present invention include oligonucleotides, enzymes, dyes, fluorescent molecules, co-enzymes enzyme substrates, radioactive groups, small organic molecules, polynucleotide sequences and solid surfaces
  • the detection method includes the detection of a difference between a target nucleic acid and a reference nucleic acid when the difference is a mutation
  • the complex includes a Hol day junction
  • a further aspect of the present invention is a kit comprising, along with standard reagents, the oligonucleotides of the present invention
  • FIGS 1a, 1 b and 2 are schematic diagrams depicting the method of producing multiple copies of a target RNA or single stranded DNA using a template switch ohgonucleotide according to the present invention
  • FIG 3 is a schematic diagram generally representing an example of the branch migration inhibition method of detection of a mutation in a nucleic acid sequence
  • FIGS 4a and 4b a schematic diagrams depicting the amplification method of the present invention using labeled primers and primers with ohgonucleotide tails according to the present invention
  • FIG 5 is a schematic diagram depicting the double stranded DNA substrates (partial duplexes) for forming the signal generating cruciform structures of the present invention
  • FIG 6 is a schematic diagram depicting generation of the signal generating cruciform structures of the present invention
  • FIG 7 is a schematic diagram depicting the detection of the amplification products using two labeled probes
  • the present invention describes an isothermal, transcription-based nucleic acid amplification method which is based on the formation of unique target-dependent nucleic acid species by template switching This product species can be amplified further to produce both double-stranded DNA products and multiple copies of a single- stranded RNA product
  • the single-stranded RNA amplification products are of the same sense as a target sequence
  • the new amplification method of this invention provides a well-defined mechanism for DNA target sequence amplification and further requires denaturation of a double-stranded DNA target prior to the amplification of a specific sequence
  • the new method provides means for specific amplification of an RNA sequence in the presence of double-stranded DNA target
  • the new method leads to a more controlled formation of the substrate for the DNA-dependent RNA polymerase, and thus to a better control of the kinetics of amplification
  • the invention further provides a process for performing analysis of sequence alteration, or genotyping, using detection by the branch migration inhibition method as described in WO 97/23646
  • nucleic acid « a compound or composition that is a polymeric nucleotide or polynucleotide
  • the nucleic acids include both nucleic acids and fragments thereof from any source, in purified or unpu ⁇ fied form including DNA (dsDNA and ssDNA) and RNA, including t-RNA, m-RNA, r-RNA, mitochondria!
  • nucleic acid can be only a minor fraction of a complex mixture such as a biological sample
  • the nucleic acid can be obtained from a biological sample by procedures well known in the art Also included are genes, such as hemoglobin gene, cystic fibrosis gene, oncogenes, and the like Where the nucleic acid is RNA, it is first converted to cDNA by means of a primer and reverse transc ⁇ ptase
  • the nucleotide polymerase used in the present invention for carrying out amplification and chain extension can have reverse transc ⁇ ptase activity Sequences of interest may be embedded in sequences of any length of the chromosome, cDNA,
  • Chain extension of nucleic acids - extension of the 3'-end of a polynucleotide in which additional nucleotides or bases are appended
  • Chain extension relevant to the present invention is template dependent, that is, the appended nucleotides are determined by the sequence of a template nucleic acid to which the extending chain is hybridized
  • the chain extension product sequence that is produced is complementary to the template sequence
  • chain extension is catalyzed by a nucleotide polymerase
  • Target nucleic acid sequence (test nucleic acid sequence) - a sequence of nucleotides to be studied either for the presence of a difference from a related sequence or for the determination of its presence or absence
  • the target nucleic acid sequence may be double stranded or single stranded and from a natural or synthetic source
  • the target sequence usually exists within a portion or all of a nucleic acid, the identity of which is known to an extent sufficient to allow preparation of various primers necessary for introducing one or more priming sites flanking the target sequence or conducting an amplification of the target sequence or a chain extension of the products of such amplification in accordance with the present invention Accordingly, other than for the sites to which the primers bind, the identity of the target nucleic acid sequence may or may not be known In general in PCR, primers hybridize to, and are extended along (chain extended), at least the target sequence, and, thus, the target sequence acts as a template
  • the target sequence usually contains from about 30 to 20,000 or more nucleotides,
  • Reference nucleic acid sequence a nucleic acid sequence that is related to the target nucleic acid in that the two sequences are identical except for the presence of a difference, such as a mutation Where a mutation is to be detected, the reference nucleic acid sequence usually contains the normal or "wild type" sequence In certain situations the reference nucleic acid sequence may be part of the sample as, for example, in samples from tumors the identification of partially mutated microorganisms, or identification of heterozygous carriers of a mutation
  • the identity of the reference nucleic acid sequence need be known only to an extent sufficient to allow preparation of various primers necessary for introducing one or more priming sites flanking the reference sequence or conducting an amplification of the target sequence or a chain extension of the products of such amplification in accordance with the present invention Accordingly, other than for the sites to which the primers bind, the identity of the reference nucleic acid sequence may or may not be known
  • the reference nucleic acid sequence may be a reagent employed in the methods in accordance with the present invention Depending on the method of preparation of this reagent it may or may not be necessary to know the identity of the reference nucleic acid
  • the reference nucleic acid reagent may be obtained form a natural source or prepared by known methods such as those described below in the definition of oligonucleotides Holhday junction -- the branch point in a four way junction in a complex of two nucleic acid
  • Mutation a change in the sequence of nucleotides of a normally conserved nucleic acid sequence resulting in the formation of a mutant as differentiated from the normal (unaltered) or wild type sequence Mutations can generally be divided into two general classes, namely, base-pair substitutions and frameshift mutations The latter entail the insertion or deletion of one to several nucleotide pairs A difference of one nucleotide can be significant as to phenotypic normality or abnormality as in the case of, for example, sickle cell anemia
  • Duplex a double stranded nucleic acid sequence wherein all, or substantially all, of the nucleotides therein are complementary
  • Ohgonucleotide - a single stranded polynucleotide, usually a synthetic polynucleotide
  • the ohgonucleot ⁇ de(s) are usually comprised of a sequence of 10 to 100 nucleotides, preferably, 20 to 80 nucleotides in length
  • ohgonucleotide utilized in the present invention
  • Such ohgonucleotide can be obtained by biological synthesis or by chemical synthesis
  • chemical synthesis will frequently be more economical as compared to the biological synthesis
  • chemical synthesis provides a convenient way of incorporating low molecular weight compounds and/or modified bases during the synthesis step
  • chemical synthesis is very flexible in the choice of length and region of the target polynucleotide binding sequence
  • the ohgonucleotide can be synthesized by standard methods such as those used in commercial automated nucleic acid synthesizers
  • Chemical synthesis of DNA on a suitably modified glass or resin can result in DNA covalently attached to the surface This may offer advantages in washing and sample handling
  • standard replication methods employed in molecular biology can be used such as the use of M13 for single stranded DNA as described by Messing J , Methods Enzymol, 101 20-78 (1983)
  • ohgonucleotide synthesis examples include phosphot ⁇ ester and phosphodiester methods, Narang, et al, Meth Enzymol, 68 90 (1979) and synthesis on a support, Beaucage, et al , Tetrahedron Letters, 22 1859-1862 (1981), as well as phosphoramidate technique, Caruthers, M , 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 Ohgonucleotide p ⁇ mer(s) - an ohgonucleotide that is usually employed in a chain extension on a polynucleotide template such as in, for example, an amplification of a nucleic acid
  • the ohgonucleotide primer is usually a synthetic ohgonucleotide that is single stranded,
  • the number of nucleotides in the hyb ⁇ dizable sequence of the ohgonucleotide primer will be at least ten nucleotides, preferably at least 15 nucleotides and, preferably 20 to 50, nucleotides
  • the primer may have a sequence at its 5'- end that does not hybridize to the target or reference polynucleotides that can have 1 to 60 nucleotides, preferably, 8 to 30 polynucleotides Nucleoside t ⁇ phosphates -- nudeosides having a 5'-t ⁇ phosphate substituent
  • the nudeosides are pentose sugar derivatives of nitrogenous bases of either pu ⁇ ne or py ⁇ midine derivation, covalently bonded to the 1 '-carbon of the pentose sugar, which is usually a deoxyribose or a ⁇ bose
  • the pu ⁇ e bases comprise aden ⁇ ne(A), guanine (G), inosine (I), and derivatives and analogs thereof
  • the py ⁇ midine bases comprise cytosine (C), thymine (T), uracil (U), and derivatives and analogs thereof
  • Nucleoside t ⁇ phosphates include deoxy ⁇ bonucleoside t ⁇ phosphates such as the four common t ⁇ phosphates dATP, dCTP, dGTP and dTTP and ⁇ bonucleoside t ⁇ phosphates such as the four common t ⁇ phosphates rATP, rCTP, rGTP and rUTP.
  • nucleoside t ⁇ phosphates also includes derivatives and analogs thereof, which are exemplified by those derivatives that are recognized and polymerized in a similar manner to the undenvatized nucleoside t ⁇ phosphates
  • 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 monomenc unit of nucleic acid polymers, i e , DNA and RNA
  • Nucleoside - is a base-sugar combination or a nucleotide lacking a phosphate moiety
  • the nucleotide polymerase is a template dependent polynucleotide polymerase and utilizes nucleoside t ⁇ phosphates 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, and reverse transc ⁇ ptase, and may be thermally stable DNA polymerases such as Vent® DNA polymerase, VentR® DNA polymerase, Pfu® DNA polymerase, Taq® DNA polymerase, and the like, derived from any source such as cells, bacteria, such as E coli, plants
  • 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
  • Increased stringency is achieved by elevating the temperature, increasing the ratio of cosolvents, lowering the salt concentration, and the like
  • 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 3' end of each sequence binds to the 5'-end of the other sequence and each A, T(U), G, and C of one sequence is then aligned with a T(U), A, C, and G, respectively, of the other sequence "Complementary" does not require that sequences have 100% base pairing Sequences capable of hyb ⁇ zing to each other, although having base pair mis- matches, are complementary for the purposes of this invention
  • Copy -- means a sequence that is a direct identical copy of a single stranded polynucleotide sequence as differentiated from a sequence that is complementary to the sequence of such single stranded polynucleotide
  • Conditions for extending a primer - includes a nucleotide polymerase, nucleoside t ⁇ phosphates or analogs thereof capable of acting as substrates for the polymerase and other materials and conditions required for enzyme activity such as a divalent metal ion (usually magnesium) pH, ionic strength, organic solvent (such as formamide), and the like
  • hgand and receptor members of an immunological pair such as antigen-antibody, or may be operator-repressor, nuclease-nucleotide, biotin-avidin, hormone-hormone receptor, IgG-protein A, DNA-DNA, DNA-RNA, and the like
  • Ligand any compound for which a receptor naturally exists or can be prepared
  • 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 and synthetic receptors, e g , thyroxine binding globulin, antibodies Fab fragments thereof, enzymes, lectins, nucleic acids, repressors, oligonucleotides protein A, complement component C1q, or DNA binding proteins and the like Small organic molecule - a compound of molecular weight less than about
  • the small organic molecule can provide a means for at- tachment of a nucleotide sequence to a label or to a support.
  • the support can be hydrophilic or capable of being rendered hydrophilic and includes inorganic powders such as silica, magnesium sulfate, and alumina; natural polymeric materials, particularly cellulosic materials and materials derived from cellulose, such as fiber containing papers, e.g., filter paper, chromatographic paper, etc.; synthetic or modified naturally occurring polymers, such as nitrocellulose, cellulose acetate, poly (vinyl chloride), polyacrylamide, cross linked dextran, agarose, polyacrylate, polyethylene, polypropylene, poly(4-methylbutene), polystyrene, poiymethacrylate, poly(ethylene terephthalate), nylon, poly(vinyl butyrate), etc.; either used by themselves or in conjunction with other materials; glass available as Bioglass, ceramics, metals, and the like. Natural or synthetic assemblies such as liposomes, phospholipid vesicles, and cells can also
  • 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,
  • the surface can have any one of a number of shapes, such as strip, rod, particle, including bead, and the like.
  • Labels include reporter molecules that can be detected directly by virtue of generating a signal, and specific binding pair members that may be detected indirectly by subsequent binding to a cognate that contains a reporter molecule such as ohgonucleotide sequences that can serve to bind a complementary sequence or a specific DNA binding protein; organic molecules such as biotin or digoxigenin that can bind respectively to streptavidin and anti-digoxin antibodies, respectively; polypeptides; polysaccharides; and the like. In general, any reporter molecule that is detectable can be used.
  • the reporter molecule can be isotopic or nonisotopic, usually non-isotopic, and can be a catalyst, such as an enzyme, dye, fluorescent molecule, chemiluminescer, coenzyme, enzyme substrate, radioactive group, a particle such as latex or carbon particle, metal sol, crystallite, liposome, cell, etc., which may or may not be further labeled with a dye, catalyst or other detectable group, and the like.
  • the reporter group can be a fluorescent group such as fluorescein, a chemiluminescent group such as luminol, a terbium chelator such as N-(hydroxyethyl) ethylenediaminetriacetic acid that is capable of detection by delayed fluorescence, and the like.
  • the label is a member of a signal producing system and can generate a detectable signal either alone or together with other members of the signal producing system.
  • a reporter molecule can serve as a label and can be bound directly to a nucleotide sequence.
  • the reporter molecule can bind to a nucleotide sequence by being bound to an sbp member complementary to an sbp member that comprises a label bound to a nucleotide sequence. Examples of particular labels or reporter molecules and their detection can be found in U.S. Patent 5,595,891 , 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 the label.
  • the signal producing system generates a signal that relates to the presence of the analyte or the presence of a difference between the target polynucleotide sequence and the reference polynucleotide sequence.
  • the signal producing system includes all of the reagents required to produce a measurable signal.
  • the reporter molecule is normally bound to a sbp member complementary to a sbp member that is bound to or is part of a nucleotide sequence.
  • the signal producing system can include substrates, enhancers, activators, chemiluminescent compounds, cofactors, inhibitors, scavengers, metal ions, specific binding substances required for binding of signal generating substances, coenzymes, substances that react with enzymic products, enzymes and catalysts, and the like.
  • the signal producing system provides a signal detectable by external means, such as by use of electromagnetic radiation, electrochemical detection, desirably by spectrophotometric detection.
  • buffers will normally be present in the assay medium, as well as stabilizers for the assay medium and the assay components.
  • proteins may be included, such as albumins, organic solvents such as formamide, quaternary ammonium salts, polycations such as dextran sulfate, surfactants, particularly non-ionic surfactants, binding enhancers, e g , polyalkylene glycols, or the like
  • template switching refers to switching of polymerase catalyzed primer extension from the original target template to a segment of an ohgonucleotide, a template switch ohgonucleotide, which is annealed to the target strand downstream from the primer annealing site
  • the novel amplification method of the present invention utilizes two ohgonucleotide (DNA) primers, P1 and P2, and one template switch ohgonucleotide (DNA) Primer P1 and the template switch ohgonucleotide (TSO) are able to hybridize to the same single-strand RNA or DNA target While an RNA single- stranded target is readily amplifiable by the new procedure, amplification of a double- stranded DNA target requires a denaturation step prior to amplification to yield a single- stranded target for the subsequent hybridization of P1 and the TSO
  • Primer P1 is composed of a sequence complementary to the target
  • the TSO is composed of two sections, the 3' portion, D, which is complementary to the target, and a 5'-ta ⁇ l portion
  • This 5'-port ⁇ on of the TSO is composed of three sections A, B, and C Sequence A, is complementary to sequence A' of the target complementary section of the TSO and is, in turn, the same as an A sequence of the target
  • the A' portion of the TSO is at the 5'-end of the target complementary portion, D, of the TSO
  • the two partners of this equilibrium are (1) the fully hybridized primer extension product and the partially hybridized TSO (with portion of the A' region of the target complementary portion displaced from hybridization to the target strand by the primer extension product), and (2) fully hybridized TSO and partially hybridized primer extension product, where the 3' most portion of the extending strand is not hybridized to the target strand (displace by the TSO).
  • the 3' region of the primer extension product in the last case can hybridize to the A region of the TSO.
  • primer extension product results in formation of a thermodynamically stable tri molecular complex and leads to disruption of the equilibrium described above in favor of the second partner.
  • Primer extension then proceeds along the TSO strand and template switching is accomplished.
  • Primer extension into the downstream double stranded portion usually extends to 1 to 10 bases, dependent on the sequence content and the temperature of the reaction. Thus extension into a GC rich segment is more limited than extension into an AT rich segment.
  • the A portion should comprise of about 10 nucleotides and should be moderately AT rich.
  • Sequence B of the TSO as described herein, which is immediately 5' to A, is not related to the target sequence.
  • Sequence C which is immediately 5' to sequence B, is the same as a single stranded (pro-promoter) sequence of the promoter of the DNA- dependent RNA polymerase.
  • Primer P2 is composed of sequences B and C and is identical to the B and C sequence at the 5'-end of the TSO.
  • Sequence B may be any sequence which is not related to the target and represents the most optimal sequence for the DNA-dependent RNA polymerase used. In cases when it is desired to limit the length of the B sequence, it is possible to include in the 3'-end of
  • RNA Target a few of the 5'-end residues of section A of the TSO. It is desirable to limit the number of the A nucleotides in the P2 sequence so as to make it substantially non- complementary to the target sequence. This restriction will ensure that P2 is unable to hybridize to the initial target molecule and be subsequently extended by the reverse transcriptase enzyme. Amphfication of an RNA Target
  • Either two or three enzymes are used in the amplification reaction (1) reverse transc ⁇ ptase, capable of synthesizing the complement of either an RNA or a DNA single-stranded target molecule by extending a primer hybridized to the target molecule, (2) an RNase H which degrades an RNA strand of an RNA/DNA heteroduplex, however, the RNase H activity may reside in the reverse transc ⁇ ptase enzyme or could be a separate entity, and (3) an RNA polymerase which requires a double-stranded DNA promoter sequence for production of an RNA product complementary to a DNA template molecule
  • a sample suspected of containing the specific RNA target is mixed with the appropriate buffer, primers P1 and P2 the TSO, and NTPs (dNTPs and rNTPs), as required for transcription-based amplification
  • the mixture is heated to 65°C for a short period, to allow denaturation of secondary structures in the RNA target
  • the mixture is then incubated at 41 °C (
  • the DNA-dependent RNA polymerase then binds to the double-stranded promoter sequence to transcribe the newly formed template-switch DNA product, producing a plurality of an RNA transcription product, IV, which is the same sense as the initial RNA target molecule
  • the next sequence of reactions shown in FIG 1 b results in the formation of a double-stranded DNA product, which is a substrate for the DNA-dependent RNA polymerase, to produce additional RNA products similar to the products produced in the initial sequence of reactions
  • a cycle for exponential amplification of the initial target nucleic acid is established
  • Primer P1 hybridizes to the P1' sequence at the 3'-end of the RNA product molecule, V RT then extends the primer to replicate the RNA product, resulting in formation of an RNA/DNA heteroduplex VI RNase H then degrades the RNA molecule of the heteroduplex, resulting in formation of a single- stranded DNA product VII
  • P ⁇ mer P2 hybridizes to the B' sequence at the 3'-end of the
  • DNA product to form complex VIII RT then extends P2 to replicate the DNA product
  • RT also extends the 3' end of the single-stranded DNA product to form a fully double-stranded promoter
  • the double-stranded DNA product, IX is a substrate for the DNA-dependent RNA polymerase, to produce multiple copies of the single-stranded RNA product IV
  • This last product, IV is a substrate for formation of the double-stranded DNA product, leading to exponential amplification of the target molecule
  • Amplification of a DNA Target Amplification of DNA target molecules can proceed only following denaturation of the double-stranded target This restriction makes the present invention especially useful for the amplification of RNA templates in the presence of excess genomic DNA
  • target amplification follows the hybridization of a single primer which is 5'-ta ⁇ led by the promoter sequence Hybridization of this primer to double-stranded DNA target may occur due to partial denaturation of the double-stranded target at elevated temperature, 65°C, in the presence of DMSO (included in the amplification mixture for reduction of secondary structure in the template) This process may occur without intentional denaturation of the double-stranded DNA target and will thus reduce the specificity of amplification of an RNA target in samples containing a similar target integrated in DNA molecules contained in the sample, such as genomic DNA
  • RNA gene sequence is often required for determination of either free viral components or for determination of gene expression, as defined by sequence expressed in mRNA species
  • the formation of substrate for RNA polymerase is dependent on the hybridization of both TSO and P1 to the same nucleic acid strand, and subsequent template switch in the first primer extension step
  • a sample suspected of containing the specific DNA target is mixed with the appropriate buffer, primers P1 and P2, a TSO, and NTPs (dNTPs and rNTPs), as required for transcription-based amplification
  • the mixture is heated to 95°C for a short period to allow denaturation of the double-stranded DNA target At the end of this period, the mixture is incubated at 65°C for a short period and is then incubated at 41 °C (or the temperature suitable for activity of the enzymes used for target amplification)
  • these oligonucleotides will hybridize to the target at either 65°C or 41 °C, to form complex X P1 hybridizes to the same target DNA strand as the TSO, at a position which is 3' to the TSO hybridization sequence Following a short incubation at 41 °C, the amplification enzymes are added
  • the reverse transc ⁇ ptase extends the P1 primer along the target molecule, up to the site of TSO hybridization A template switch will occur at this site, as previously described (Patel R et al , 1996, supra) Primer extension then follows along the TSO single-stranded template to produce the complement of the 5'-port ⁇ on of TSO (which is not complementary to the target) This process results in the production of three-stranded DNA structure XI, which includes a fully functional, double-stranded promoter of the RNA polymerase The efficiency of template switch to the TSO is likely to be dependent on the
  • DNA polymerase used and the nature of the target nucleic acid to be amplified In the case of DNA target sequence it is possible that a DNA dependent DNA polymerase affords a more efficient template switch than a reverse transc ⁇ ptase In this case, a DNA dependent DNA polymerase may be included in the amplification reaction mixture Various DNA dependent DNA polymerase are commercially available and are suitable for use in carrying out the present invention
  • RNA product IV The DNA-dependent RNA polymerase will then bind at the promoter site and produce multiple copies of an RNA product IV, which is the same sense as the initial target sequence
  • These products will serve as a template for formation of double- stranded DNA products which are the substrate for T7 RNA polymerase, as was described for the amplification of an RNA target
  • the process is composed of the following steps hybridization of the P1 primer to product IV to produce product V, extension of the primer by RT to form RNA/DNA heteroduplex VI, degradation of the RNA template by RNase H to yield a single-stranded DNA product VII, hybridization of primer P2 to the single-strand DNA product, and extension of P2 and the single stranded DNA product by the RT to produce a double stranded DNA product IX, which is in turn a substrate for the RNA polymerase, to produce a plurality of the single-stranded RNA product IV
  • This process results in further amplification by production of multiple copies of the sense RNA
  • the incubation temperatures are given as an example and are not limited to the exact temperatures cited The temperatures will be determined by the requirements for denaturation of the secondary structures of the specific target and the optimum temperature for the amplification enzymes Moreover, when thermostable enzymes are used, the enzymes can be included in the initial reaction mixture, thus eliminating the need for a separate addition of the amplification enzymes following the initial incubations at elevated temperatures
  • RNA products produced by amplification of either RNA or DNA target by the disclosed method are of the same sense as the target nucleic acid strand, in contrast to product of the currently known transcription-based, amplification methods such as NASBA and TMA
  • the restrictive requirement of formation of a t ⁇ -molecular complex, which serves as a unique substrate for template switch during the first primer extension step results in high specificity of RNA target amplification in the presence of excess double stranded DNA target
  • RNA targets usually mRNA
  • the amplification primers and TSO are desirable to be complementary to sequences of the DNA target strand which are not included in the mRNA
  • the amplification of a DNA sequence which spans noncodmg sequences, i e are not present in mRNA, such as intron sequences, may also serve to enhance the specificity of amplification of a DNA sequence
  • RNA products renders the amplification process suitable for a wide range of detection processes, including solution phase homogeneous detection methods, solid phase-based methods, and the various array- based methods
  • BMI Branch Migration Inhibition
  • the present invention includes a novel scheme for the formation of substrates for BMI detection of sequence alterations employing the disclosed strand switch isothermal nucleic acid amplification
  • the method is applicable for detection of sequence alteration in either RNA or DNA target sequences, as required for different applications
  • the in vivo production of multiple copy of mRNA increases the amount of target molecules which in turn reduces the level of in vitro amplification required for subsequent analysis
  • the target molecule for analysis will be DNA
  • the BMI method for detection of sequence alteration requires the formation of amplification products which are capable, upon denaturation and re-association, of forming partial duplex
  • the production of amplification products capable of forming the required partial duplexes is made possible by the use of a mixture of a forward primer P2, and two reverse primers, P1 and P3, for the amplification of both a test and a reference DNA sequence
  • the two reverse primers have a common 3'-port ⁇ on, Pa, which is complementary to the target, and 5'-ta ⁇ l sequences, A1 and B1 , which are different for the two reverse primers and are not related to the target
  • the tailed duplexes form a quadromolecular complex If a difference exists between the test sequence and the reference sequence, as depicted in FIG 3 as M, strand exchange in the complex ceases, resulting in the formation of a stable complex, C
  • This design is adapted for the template switch isothermal amplification by the modification of the modification of the
  • FIG 4a depicts the amplification of either the test or the reference sequence, as only one sequence is shown However it should be understood that the amplification scheme is identical for both the test and reference nucleic acid sequences It is preferred that the amplification of the test and reference sequences take place in one vessel However, amplification in separate vessels is contemplated as part of the present invention
  • the mixture is heated to 95°C for denaturation of a DNA target
  • the mixture is then incubated briefly at 65°C, followed by brief incubation at 41 °C, as previously described herein
  • the initial incubation at 95°C is omitted
  • a mixture of the amplification enzymes is then added to the reaction mixture and target amplification proceeds at the same temperature
  • Amplification of a DNA target is initiated by formation of t ⁇ molecular complexes XII (test or reference, TSO and F1) and XIII (test or reference, TSO, and
  • Primer extension by RT and template switch proceed as previously described herein to produce the three-stranded DNA structures XIV and XV, which have a double- stranded promoter for the RNA polymerase
  • the RNA polymerase produces multiple copies of an RNA transcript, XVI and XVII, of the DNA strands formed by primer extension and template switch
  • Primers F1 and F2 hybridize to the respective RNA products to yield complexes XVIII and XIX
  • RNA/DNA heteroduplexes XX and XXI results in formation of RNA/DNA heteroduplexes XX and XXI
  • the RNA strand of these heteroduplexes is then degraded by RNase H to yield single-strand DNA products XXII and XXIII
  • the 5' biotin or digoxigenin-labeled primers P2 hybridize to the single-stranded DNA products
  • RNA polymerase binds to the double-stranded products at the promoter site and produces multiple copies of RNA transcripts XXVI and XXVII These products serve as substrates for formation of double-stranded products as described before
  • the double- stranded DNA products biotin-or dig -labeled XXIV and biotin- or dig -labeled XXV are suitable substrates for BMI analysis
  • RNA molecule products XIV and XV are composed of both DNA and RNA strands as was previously described for the amplification of an RNA target The RNA strand of these products is degraded by
  • the product contains a double-stranded promoter for the RNA polymerase Further amplification of the RNA target sequence proceeds as described above for a DNA target sequence
  • partial duplexes comprise double stranded nucleic acid sequences wherein one end thereof has non- complementary ohgonucleotide sequences, one linked to each strand of the double stranded molecule
  • Each non-complementary sequence has 8 to 60, preferably, 10 to 50, more preferably, 15 to 40, nucleotides
  • the degradation of the RNA products prior to BMI analysis is required since these products can compete with the DNA products, as follows
  • Second, the single-stranded RNA products have a t1' or t2' sequence at their 3'-end, which can compete with the annealing of the partial DNA duplexes, if formed, to form the four-stranded DNA cruciform structures
  • FIG 6 the association of the partial duplexes by hybridization of the respective tail sequences forms four-stranded cruciform structures capable of strand exchange via spontaneous branch migration
  • branch migration results in strand exchange, formation of fully double-stranded DNA duplexes, and dissociation of the two labels
  • test sequence When the test sequence is not identical to the reference sequence branch migration is inhibited, resulting in the formation of stable, four-stranded cruciform structures which are labeled by the two labels These stable cruciform structures are detectable
  • Detection of the stable cruciform structures can be carried out using a variety of methods suitable for the detection of the association of the two labels
  • detection can be carried out by EIA EIA using streptavidin coated microtiter plates and an enzyme anti digoxin monoclonal antibody conjugate as previously described in Lishanski et al 1996, A homogenous mutation detection method based on inhibition of branch migration, Abstract of the 28 th Annual
  • Homogeneous detection methods are preferred Various homogeneous detection methods are known, such as the scintillation proximity assay method the electrochemical luminescence method, and the Luminescent Oxygen Channeling Immunoassay (LOCI) detection method
  • Nonspecific PCR priming was shown to lead to formation of stable cruciform structures which are independent of sequence alteration
  • misp ⁇ ming in the current amplification method cannot lead to the formation of amplification products, in so far as products of misp ⁇ ming are not expected to undergo the switch of replication template from the target to the TSO
  • the last step is essential for the formation of the first double-stranded functional promoter for the RNA polymerase No amplification is possible when this unique product is not formed
  • an aqueous medium is employed
  • Other polar cosolvents may also be employed, usually oxygenated organic solvents of from 1 to 6, more usually from 1 to 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 - 8 5
  • the pH and temperature are chosen and varied, as the case may be, so as to cause, either simultaneously or sequentially, dissociation of any internally hybridized sequences, hybridization of the ohgonucleotide primers or TSO, with the target nucleic acid sequence, extension of the primers
  • Various buffers may be used to achieve the desired pH and maintain the pH during the determination
  • Illustrative buffers include borate, phosphate, carbonate,
  • the particular buffer employed is not critical to this invention but in individual methods one buffer may be preferred over another
  • the buffer employed in the present methods normally contains magnesium ion (Mg 2+ ), which is commonly used with many known polymerases, although other metal ions such as manganese have also been used
  • magnesium ion is used at a concentration of from about 1 to 20mM, preferably, from about 1 5 to 15mM, more preferably, 3-12mM
  • the magnesium can be provided as a salt, for example, magnesium chloride and the like
  • the primary consideration is that the metal ion permit the distinction between different nucleic acids in accordance with the present invention
  • the concentration of the nucleotide polymerase is usually determined empirically Preferably, a concentration is used that is sufficient such that further increase in the concentration does not decrease the time for the amplification by over 5-fold, preferably 2-fold
  • the primary limiting factor generally is the cost of the reagent
  • the amount of the target nucleic acid sequence to be subjected to subsequent amplification using primers in accordance with the present invention may vary from about 1 to 1010, more usually from about 103 to 108 molecules, preferably at least 10- 21M in the medium and may be 10-10 to 10-19M, more usually 10-14 to 10-19M
  • the amount of the ohgonucleotide primers and TSO used in the amplification reaction in the present invention will be at least as great as the number of copies desired and will usually be 10-9 to 10-3 , preferably, 10-7 to 10-4 M
  • the concentration of the ohgonucleotide p ⁇ mer(s) is substantially in excess over, preferably at least 100 times greater than, more preferably, at least 1000 times greater than, the concentration of the target nucleic acid sequence
  • concentration of the nucleoside t ⁇ phosphates in the medium can vary widely, preferably, these reagents are present in an excess amount for both amplification and chain extension
  • the nucleoside t ⁇ phosphates are usually present in 10-6 to 10-2M, preferably 10-5 to 10-3M
  • the identity of the target nucleic acid sequence does not need to be known except to the extent to allow preparation of the necessary p ⁇ mers and TSO for carrying out the above reactions
  • the present invention permits the determination of the presence or absence of a mutation in
  • Sequence determination could be directly obtained from the amplification products generated by the disclosed method
  • Various methods could be employed for sequence determination Transcription-based sequencing (Sasaki N , et al, PNAS, 95 3455-3460 (1998)) can be carried out using the double-stranded DNA product as a substrate
  • sequencing by hybridization of the single-stranded RNA product to an ohgonucleotide array (Gene Chip) could be employed
  • Other methods which are based on probe hybridization to the single-stranded RNA product could also be used for obtaining sequence information
  • the combination of BMI analysis for detection of gene sequence alteration and sequence determination of altered test sequences is most desirable for large-scale testing, such as in screening for genetic abnormalities in which the abnormal state is associated with various gene alterations of a given sequence, as well as other life science applications requiring the assessment of sequence identity
  • the combination of BMI analysis for detection of gene sequence alteration and sequence determination of altered test sequences is most desirable for large-scale testing, such as in screening for genetic abnormal
  • One means of detecting the RNA product generated by the amplification of the present invention is formation of a t ⁇ molecular complex comprising the single stranded
  • RNA product and two ohgonucleotide probes Each of the ohgonucleotide probes comprises a sequence, which is complementary to a sequence on the RNA product, and a first or second labels, respectively.
  • the two labels become associated by virtue of both being present within the t ⁇ molecular complex, in the presence of the single stranded amplification products Detection of the association of the two labels in the complex provides for detection of the complex and thus detection of the amplification product
  • one means of detecting the stable cruciform structures, indicating sequence alteration in a test sequence relative to a reference sequence involves the use of two labels on non-complementary strands of the quadramolecular complex
  • the two labels become associated by virtue of both being present in the quadamolecular complex if a difference is present between the related sequences
  • Detection of the two labels in the complex provides for detection of the complex and thus detection of the presence of difference between the two related sequences
  • the association of the two labels within the complex is detected
  • Detection of the association of two labels in a stable complex provides for detection of either the t ⁇ molecular complex or the quadramolecular complexes of the present invention
  • the association of the labels within the complex may be detected in many ways
  • one of the labels can be an sbp member and a complementary sbp member is provided attached to a support Upon the binding of the complementary sbp members to one another, the complex becomes bound to the support and is separated from the reaction medium
  • the other label employed is a reporter molecule that is then detected on the support
  • the presence of the reporter molecule on the support indicates the presence of the complex on the support, which in turn indicates the presence of the mutation in the target nucleic acid sequence
  • ELISA enzyme-linked immunosorbent assay
  • the sbp member is biotin
  • the complementary sbp member is streptavidin
  • the reporter molecule is an enzyme such as alkaline phosphatase Detection of the signal will depend upon the nature of the signal producing system utilized If the reporter molecule is an enzyme, additional members of the
  • the association of the labels within the complex may also be determined by using labels that provide a signal only if the labels become part of the complex 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 accomplished by employing at least one suspendable particle as a support, which may be bound directly to a nucleic acid strand or may be bound to an sbp member that is complementary to an sbp member attached to a nucleic acid strand
  • Such a particle serves as a means of segregating the bound target polynucleotide sequence from the bulk solution, for example, by settling, electrophoretic separation or magnetic separation
  • a second label, which becomes part of the complex is a part of the signal producing system that is separated or concentrated in a small region of the solution to facilitate detection Typical
  • 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 elevated temperatures used for amplification, chain extension and branch migration
  • polynucleotide or polynucleotide analogs having 5 to 20 or more nucleotides depending on the nucleotides used and the nature of the analog
  • Polynucleotide analogs include structures such as poly ⁇ bonucleotides, polynucleoside phosphonates, peptido-nucleic acids, polynucleoside phosphorothioates, homo DNA
  • oligonucleotides or polynucleotide analogs that have sequences of nucleotides that are complementary to the label sequences
  • One of these oligonucleotides or ohgonucleotide analogs is attached to, for example, a reporter molecule or a particle
  • the other is attached to a primer, either primer F1 or primer F2 and/or P2 or a probe, as a label
  • Neither the ohgonucleotide nor polynucleotide analog attached to the primers should serve as a polynucleotide polymerase template This is achieved by using either a polynucleotide analog or a polynucleotide that is connected to the primer by an abasic group
  • the abasic group comprises a chain of 1 to 20 or more atoms, preferably at least 6 atoms, more preferably, 6 to 12 atoms
  • an ohgonucleotide or polynucleotide analog attached to a reporter molecule or a particle can bind to its complementary polynucleotide analog or ohgonucleotide separated by an abasic site that has become incorporated into the partial duplexes as labels during amplification If the oligonucleotides or polynucleotides analog become part of a t ⁇ molecular or quadramolecular complex, the reporter molecule or particle becomes part of the complex By using different polynucleotide analogs or ohgonucleotide sequences for labels, two different reporter molecules or particles can become part of the complex Various combinations of particles and reporter molecules can be used
  • the polynucleotide analog or ohgonucleotide label is attached to a probe, as used for the detection of a single stranded amplification product
  • the polynucleotide analog of ohgonucleotide are attached directly at the 5' or the 3' end of the probe sequence which is complementary to the target In so far as the probes do not serve as substrates for target dependent extension, the attachment of the label sequence to the probe using an abasic spacer is not required Under proper annealing conditions the labeled probes hybridize to the single stranded amplification product to form a stable complex
  • detection of the single stranded product is carried out using two labeled probes, a t ⁇ molecular complex is formed, and the two polynucleotide analog or ohgonucleotide labels, each attached to the corresponding probe, become associated within the complex
  • the single stranded amplification product can be detected by using one labeled probe and one labeled
  • one means of detecting the presence of specific nucleic acid sequence involves the isothermal amplification of the invention and detection of the single stranded RNA amplification product
  • two ohgonucleotide probes, probe 1 and probe 2, and two signal generating particles, a first signal generation particle and a second signal generating particle are employed for detection of the RNA product
  • the first probe comprises an ohgonucleotide sequence PS1 which is complementary to sequence TS1 on the RNA product and a sequence L1 which is a label and is not complementary to the sequence of the RNA product
  • the second probe comprises an ohgonucleotide sequence PS2 which is complementary to sequence TS2 on the RNA product and a sequence L2 which is a label and is not complementary to the RNA product
  • Ohgonucleotide L1', which is complementary to the label sequence L1 is attached to the first signal generating particle, which may be a chemiluminescer particle Ohgonucleotide L2'
  • an ohgonucleotide or a polynucleotide analog attached to a reporter molecule or a particle can bind to its complementary ohgonucleotide or polynucleotide analog attached to the two probes, or to one probe and one primer, as used for detection of the single stranded amplification product If the ohgonucleotide or polynucleotide analog labels become part of the complex, the reporter molecule or particle becomes part of the complex.
  • two different reporter groups or particles can become part of the complex
  • the t ⁇ molecular complexes or the quadramolecular complexes can also be detected using other hgand/receptor combinations
  • the two labels attached to the primers or probes may be small molecules such as biotin and digoxigenin and the two corresponding receptors can
  • 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 t ⁇ tiated reporter molecule and a particle containing a scmtillator
  • 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 Patent No 5,536,834, which disclosure is incorporated herein by reference It is also possible to detect particle agglutination due to binding of the two particle species as described in
  • detection of the 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
  • a particle is employed, which comprises the chemiluminescent compound associated therewith such as by incorporation therein or attachment thereto
  • the particles have a recognition sequence, usually an ohgonucleotide or polynucleotide analog, attached thereto with a complementary sequence incorporated into one of the nucleic acid strands as a first label
  • Another particle is employed that has the photosensitizer associated therewith
  • the medium is irradiated with light to excite the photosensitizer, which is capable in its excited state of activating oxygen to a singlet state
  • the chemiluminescent compound of one of the sets of particles is now in close proximity to the photosensitizer by virtue of the association of the two labels, the chemiluminescent compound is activated by the singlet oxygen and emits luminescence
  • the medium is then examined for the presence and/or the amount of luminescence or light emitted, the presence thereof being related to the presence of a stable complex
  • the presence of the latter indicates the presence and/or amount of the target polynucleotide sequence or the presence of sequence alteration in the target polynucleotide relative to a reference sequence
  • predetermined amounts of rea indicates the presence and/or amount of the target polynucleotide sequence or the presence of sequence alteration in the target polynucleotide relative to a reference sequence
  • E coh DNA target was purchased from Sigma and M tuberculosis DNA target was obtained from Stanford Medical Center All the ohgodeoxynbonudeotides used were synthesized with specific modifications by
  • RNA guard purchased from Pharmacia Biotech (Piscataway, NJ) Ultrapure nucleoside 5'-tr ⁇ phosphate (rNTP) and 2'deoxynucleos ⁇ de 5'-t ⁇ phosphates
  • dNTPs were purchased as 100 mM solution from Pharmacia Biotech (Piscataway,
  • Bovine Serum Albumin was purchased from Gibco, Lifetech (Gaithersburg, MD)
  • RNAse /DNAse-free water from Ambion (Austin, TX) PCR tubes were purchased from Corning and ISC Bioexpress (Kaysville, UT)
  • precast polyacrylamide (native) gels were purchased from Novex (San Diego,
  • Buffer A 10mM T ⁇ s-HCI, pH 8 3, 50mM KCl, 1 5mM MgCI 2 , and 200 ⁇ g/ml BSA (used for PCR amplification reactions)
  • Buffer B 40mM T ⁇ s-HCI, pH 8 5, 5mM DTT, 12mM MgCI 2 , 70mM KCl, 108 8 ⁇ g/ml
  • Buffer C 50 mM KCl , 4 mM MgCI 2 , 10 mM Tris-HCl pH 8 3 , 200 ⁇ g/ml BSA (used for
  • Hydroxypropylaminodextran (1 NH 2 / 7 glucose) was prepared by dissolving Dextran T-500 (Pharmacia, Uppsala,
  • the chlorodextran product was dissolved in 200mL of water and added to 2L of concentrated aqueous ammonia (36%) This solution was stirred for 4 days at room temperature, then concentrated to about 190mL on a rotary evaporator The concentrate was divided into two equal batches, and each batch was precipitated by pouring slowly into 2L of rapidly stirring methanol The final product was recovered by filtration and dried under vacuum
  • Hydroxypropylaminodextran (1 NH 2 / 7 glucose), prepared above, was dissolved in 50mM MOPS, pH 7 2, at 12 5 mg/mL The solution was stirred for 8 hr at room temperature, stored under refrigeration and cent ⁇ fuged for 45 m at 15,000 rpm in a Sorvall RC-5B centrifuge immediately before use to remove a trace of solid material
  • Chemiluminescer particles (TAR beads)
  • the following dye composition was employed 20% C-28 thioxene (prepared as described above), 1 6%1-chloro-9,10-b ⁇ s(phenylethynyl)anthracene (1-CI-BPEA) (from Aldrich Chemical Company) and 2 7% rubrene (from (from Aldrich Chemical Company)
  • the particles were latex particles (Seradyn Particle Technology, Indianapolis IN)
  • the dye composition (240-250 mM C-28 thioxene, 8-16 mM 1-CI- BPEA, and 20-30 mM rubrene) was incorporated into the latex beads in a manner similar to that described in U S Patent 5,340,716 issued August 23, 1994 (the '716 patent), at column 48, lines 24-45, which is incorporated herein
  • the ohgonucleotide was immobilized on the surface of the above particles in the following manner
  • Aminodextran 500 mg was partially maleimidated by reacting it with sulfo-SMCC (157 mg, 10 mL H 2 O)
  • the sulfo-SMCC was added to a solution of the aminodextran (in 40 mL, 0 05 M Na 2 HPO 4 , pH 7 5) and the resulting mixture was incubated for 1 5 hr
  • the reaction mixture was then dialyzed against MES/NaCI (2x2L, 10 mM MES, 10 mM NaCI, pH 6 0, 4°C)
  • the maleimidated dextran was cent ⁇ fuged at 15,000 rpm for 15 minutes and the supernatant collected
  • the supernatant dextran solution 54 mL was then treated with imidazole (7 mL of 1 0 M solution) in MES buffer
  • the sensitizer beads were prepared placing 600 mL of carboxylate modified beads (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 m Sihcon tetra-t-butyl phthalocyanme (10 0 g) was dissolved in 300 mL of benzyl alcohol at
  • Ohgonucleotide bound sensitizer particles were prepared in a manner similar to that described above for ohgonucleotide bound to chemiluminescer particles
  • Probe 1 and probe 2 comprise a 5 sequence which is complementary to two site on the product, PS1 of probe 1 is complementary to sequence TS1 on the RNA product and sequence PS2 of probe 2 is complementary to sequence TS2 on the RNA product
  • the two probes further comprise a label L1 and L2, respectively, at their 5' or 3' end
  • Labels L1 and L2 may be reporter molecules such as biotin and digoxigenin, or may comprise a nucleic acid sequences which are not related to the amplification product or the target
  • the t ⁇ molecular complex formed by the hybridization of the RNA product and the two probes is detectable by numerous method known in the art depending upon the labels chosen
  • FIG 7 depicts the formation of a complex between the t ⁇ molecular complex and two signal generating particles for example a chemiluminescer particle and a sensitizer particle as used in LOCI
  • the L1 and L2 labels are ohgonucleotide re ⁇
  • ohgonucleotide L1 ' which comprises a sequence complementary to L1 is attached to the first particle
  • ohgonucleotide L2 which comprises a sequence complementary to L2 is attached to the second particle
  • the signal-generating complex is formed by hybridization of the two probes to the RNA product and binding of the particles to the complex by hybridization of L1 to L1 ' and L2 to L2'
  • 13 0 ⁇ l of the reaction mixture (containing 1 mM of each dNTPs and 2 mM each of rATP, rUTP, rCTP and 1 5 mM rGTP, 0 5 mM rlTP, 250 nM of primer 1 and 2 and 50 nM of TSO DMSO 15% (U/U), in Buffer B) was ahquoted to individual PCR tubes in an 8 tube-strip 2 ⁇ l sample containing E coh genomic DNA was then added to each tube, followed by 15 ⁇ l mineral oil
  • the mixture was incubated at 95°C for 5 mm , cooled down to 41 °C and incubated for 5 mm 5 0 ⁇ l enzyme mixture (RT 6 4 u, T7 RNA polymerase 32 u, RNase H 0 08 u, and RNA guard 12 u, in 30% glycerol (v/v) in 10 mM T ⁇ s pH 8 5) was then added to each tube and the reactions were further incubated at 41 °C for 90 mm
  • LOCI detection of the amplification products was carried out as follows 45 ⁇ l of the combined detection reagents (containing 12 5 nM of probe 1 and probe 2, and 2 5 ug of the acceptor and sensitizer ohgonucleotide coated LOCI beads in Buffer C) were added to individual tubes and 5 ⁇ l amplification products (amplification reaction mixture with or without target) or water were then added The detection reaction mixtures were incubated at 65°C for 2 mm, 50°C for 15 mm and 37°C for 30 mm
  • the detection limit for amplification of the DNAJ gene sequence using RT, T7 DNA dependent RNA polymerase and RNase H is about 1000 molecules per reaction It is likely that further optimization of this amplification scheme could enhance sensitivity limit Optimization of this procedure might be achieved by selecting a DNA polymerase which provides high efficiency of template switching, as shown in the following example
  • Reaction mixtures were assembled as described in Example 2 The reaction mixtures were incubated at 95°C for 4 mm cooled down to 65°C and incubated for 2 mm Pfu polymerase (1 u) was added and the reaction mixture was further incubated at this temperature for 5 mm The reactions mixture were then cooled down to 41°C, and 5 ul of the enzyme mixture (as in Example 1) was added The reaction mixtures were further incubated as in Example 1
  • Reaction mixtures were assembled as described in Example 1 The reaction mixtures were incubated at 95°C for 4 mm , cooled down to either 50°C or 41°C and incubated for 2 mm Pfu polymerase (1 u) was added to the reaction mixture and the mixtures were incubated for 5 minutes When Pfu polymerase was added at 50°C, the reaction mixtures were cooled to 41 °C and incubated for 5 mm , before addition of 5 ul of the enzyme reaction mixture as in Example 1 When Pfu polymerase was added at 41°C, the enzyme reaction mixture was add after 5 mm incubation at this temperature In another case Pfu polymerase was mixed with the enzyme reaction mixture and the reactions were carried out as in Example 1 LOCI detection of the amplification products was the same as in previous examples
  • the efficiency of NASA amplification of DNA nucleic acid targets is improved when the first primer extension and target switch step is carried out by Pfu DNA polymerase at either 50°C or 41 °C , and Pfu polymerase may be added to the amplification enzyme mixture as a single reagent Detection limit of 10 to 100 molecules was successfully demonstrated using the two genomic DNA targets

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Abstract

L'invention concerne une méthode d'amplification isothermique d'acides nucléiques reposant sur la transcription à partir de la formation d'une espèce d'acides nucléiques dépendants de la cible par commutation de la matrice. Cette espèce de produit peut être, en outre, amplifiée pour produire des copies multiples des produits d'ADN à deux brins et de produit d'ARN à un brin. Les produits d'amplification d'ARN à un brin ont le même sens que la séquence cible. Cette invention concerne également une procédure d'inhibition reposant sur la migration de ramifications destinée à balayer des séquences d'acides nucléiques au moyen de l'amplification isothermique de commutation de brins. Un oligonucléotide de commutation de matrice utilisé dans l'amplification comprend une région 3' permettant l'hybridation d'une séquence cible et d'une région 5' qui ne s'hybride pas à la cible. La région 5' comprend une séquence promotrice d'ADN dépendant de la polymérase d'ARN. Des première et seconde amorces peuvent être utilisées pendant l'amplification et la détection. La première amorce peut permettre l'hybridation de la cible et la seconde amorce ne dépend pas de la cible. En vue de la détection, la première ou la seconde amorce a une étiquette.
PCT/US2000/013526 1999-05-17 2000-05-16 Amplification isothermique homogene et detection d'acides nucleiques utilisant un oligonucleotide de commutation de matrice Ceased WO2000070095A2 (fr)

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