WO2000070095A2 - Homogeneous isothermal amplification and detection of nucleic acids using a template switch oligonucleotide - Google Patents

Homogeneous isothermal amplification and detection of nucleic acids using a template switch oligonucleotide Download PDF

Info

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
Authority
WO
WIPO (PCT)
Prior art keywords
sequence
target
primer
dna
nucleic acid
Prior art date
Application number
PCT/US2000/013526
Other languages
French (fr)
Other versions
WO2000070095A3 (en
Inventor
Nurith Kurn
Yen Ping Liu
Original Assignee
Dade Behring Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dade Behring Inc. filed Critical Dade Behring Inc.
Publication of WO2000070095A2 publication Critical patent/WO2000070095A2/en
Publication of WO2000070095A3 publication Critical patent/WO2000070095A3/en

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813Hybridisation assays
    • C12Q1/6827Hybridisation assays for detection of mutation or polymorphism
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813Hybridisation assays
    • C12Q1/6816Hybridisation assays characterised by the detection means
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844Nucleic acid amplification reactions
    • C12Q1/6865Promoter-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

Abstract

An isothermal, transcription-based nucleic acid amplification method based on the formation of a target-dependent nucleic acid species by template switching. This product species can be amplified further to produce multiple copies of both double stranded DNA products and single stranded RNA product. The single stranded RNA amplification products are the same sense as the target sequence. Also provided is a branch migration inhibition based procedure for scanning nucleic acid sequences using the strand switch isothermal amplification. A template switch oligonucleotide used in the amplification includes a 3' region capable of hybridizing to a target sequence and a 5' region which does not hybridize to the target. The 5' region includes a promoter sequence of DNA dependent RNA polymerase. A first primer and a second primer may be used during amplification and detection. The first primer is capable of hybridizing to the target and the second primer is not target dependent. For detection, either the first primer or the second primer has a label.

Description

HOMOGENEOUS ISOTHERMAL AMPLIFICATION AND DETECTION OF NUCLEIC ACIDS USING A TEMPLATE SWITCH OLIGONUCLEOTIDE
FIELD OF THE INVENTION
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.
BACKGROUND OF THE INVENTION Significant morbidity and mortality are associated with infectious diseases. More rapid and accurate diagnostic methods are required for better monitoring and treatment of disease. Molecular methods using DNA probes, nucleic acid hybridizations and in vitro amplification techniques are promising methods offering advantages to conventional methods used for patient diagnoses.
Nucleic acid hybridization has been employed for investigating the identity and establishing the presence of nucleic acids. Hybridization is based on complementary base pairing. When complementary single stranded nucleic acids are incubated together, the complementary base sequences pair to form double stranded hybrid molecules. The ability of single stranded deoxyribonucleic acid (ssDNA) or ribonucleic acid (RNA) to form a hydrogen bonded structure with a complementary nucleic acid sequence has been employed as an analytical tool in molecular biology research. The availability of radioactive nucleoside thphosphates of high specific activity and the 32P labeling of DNA with T4 polynucleotide kinase has made it possible to identify, isolate, and characterize various nucleic acid sequences of biological interest.
Nucleic acid hybridization has great potential in diagnosing disease states associated with unique nucleic acid sequences. These unique nucleic acid sequences may result from genetic or environmental change in DNA by insertions, deletions, point mutations, or by acquiring foreign DNA or RNA by means of infection by bacteria, molds, fungi, and viruses. Nucleic acid hybridization has, until now, been employed primarily in academic and industrial molecular biology laboratories. The application of nucleic acid hybridization as a diagnostic tool in clinical medicine is limited because of the frequently very low concentrations of disease related DNA or RNA present in a patient's body fluid and the unavailability of a sufficiently sensitive method of nucleic acid hybridization analysis
One method for detecting specific nucleic acid sequences generally involves immobilization of the target nucleic acid on a solid support such as nitrocellulose 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 unhybπdized probe The support is then dried and the hybridized material is detected by autoradiography or by spectrometπc 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
Various in vitro nucleic acid amplification procedures have been described in recent years Some of the amplification methods are dependent on thermal cycling (PCR, LCR, and SPA) and others are isothermal (NASBA, TMA, Q beta rephcase and
SDA) Among the advantages of isothermal amplification are speed, simplification of instrumentation, and ease of integration with fully automated analyzers NASBA, TMA, and other variations of transcription-based amplification procedures afford amplification of single-stranded nucleic acid target molecules to produce multiple copies of a single- stranded RNA product However, these methods utilize two primers which are complementary to opposite sense, single-stranded target or product The use of the two primers of opposite polarity impairs the ability to selectively amplify a specific strand of a nucleic acid target
Another drawback of the current transcription-based, isothermal, amplification methods is the lack of control of initiation of the amplification process Amplification of an RNA target is initiated by hybridization of a primer which is 5'-taιled by a sequence representing one strand of a DNA-dependent RNA polymerase promoter The hybridization complex serves as a priming complex for a reverse transcπptase 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 acid when present in very low concentration Also, the exact mechanism for the amplification of either double-stranded DNA target molecules or a single-stranded DNA target using the current isothermal transcription-based amplification methods is not yet definitely determined This impairs the ability to specifically amplify RNA target in the presence of excess DNA The specific amplification of an RNA target is required for the detection and quantification of mRNA, as is required for analysis of gene expression, as well as the detection of free
RNA viruses in the presence of host cells which are likely to contain integrated viral genes
Regardless of the amplification used, the amplified product must be detected One method for detecting nucleic acids is to employ 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
Also, PCT application WO 97/23646, incorporated fully herein by reference, 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 When the four-stranded cruciform structures are formed in a mixture of test and reference amplification products, strand exchange by spontaneous branch migration proceeds if the test and reference amplification products are identical When the test sequence is different from the reference, branch migration is inhibited, resulting in the formation of stable, detectable four stranded cruciform structures
The recent advances in gene analysis methodology created the need for rapid, high-throughput methods for identification and quantification of specific nucleic acid sequences as well as detection of sequence alterations and the sequence determination where alteration is detected It is desirable to have a sensitive, simple method for amplifying and detecting nucleic acids, preferably in an isothermal and homogeneous format The method should minimize the number and complexity of steps and reagents Also, such method should have ability to selectively amplify a specific strand of a nucleic acid target as well as control the rate of initiation of this process Further, the ability to specifically amplify RNA target in the presence of excess DNA is highly desirable
Description of the Related Art The polymerase chain reaction (PCR) was described by Saiki, R et al,
Enzymatic Amplification of β-Globin Genomic Sequences and Restπciton Site Analysis for Diagnosis of Sickle Cell Anemia Science, 230 1350-1354 (1985), Mullis, K et al, Specific Synthesis of DNA In Vitro Via A Polymerase-Catalyzed Chain Reaction, Methods in Enzymology, 155 335-350 (1987) and U S Patent Nos 4,683,195, 4,683,202, 04,800,159, 4,465,188 and 5,008,182
Ligase Chain Reaction (LCR) 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
Figure imgf000006_0001
Nucleic acid amplification using single polynucleotide primer (ASPP) 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
Homogeneous amplification and detection of nucleic acids in a sealed tube is described in WO 97/23547
The mechanism for template switch amplification is described in Patel, R et al, Formation of chimeπc DNA primer extension products by template switching onto an annealed downstream ohgonucleotide Proc Natl Acad Sα USA, 93 2969-2974 (1996) A method of introducing defined sequences at the 3' end of a polynucleotide using a template switch ohgonucleotide is described in U S Patent No 5,679,512
Amplification of specific RNA using RNA directed polymerase such as Q beta replicase, is described in U S Patent No 4,786,600
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)
The 3SR transcription based amplification (equivalent to NASBA) is described in Guatel , J et al, Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication, Proc Natl Acad Sci USA, 87 1874-1878 (1990) Early description of amplification schemes using a primer containing a promoter sequence is provided in PCT WO 89/01050
Another isothermal amplification method, Strand Displacement Amplification, is described by Fraiser, et al, in U S Patent No 5,648,211 The method is also described in Walker, G et al , Isothermal in vitro amplification of DNA by a restriction enzyme/DNA polymerase system, Proc Natl Acad Sci USA, 89 392-396 (1992)
Chemiluminscece based homogenous detection of single standed products, Hybridization Protection Assay or HPA, is described in PCT US88/02746
Chemiluminescence based detection of SDA products is described in Walker et al , Mol Methods Virus Detect , Publisher Academic, San Diego CA, Conference, 329- 349 (1995)
Another technique for homogenous detection of single stranded amplification products using fluorescence correlation spectroscopy is described in Oehlenschlager, F et al, Detection of HIV-1 RNA by nucleic acid sequence-based amplification combined with fluorescence correlation spectroscopy, Proc Natl Acad Sci USA, 93 12811-12816 (1996)
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)
Rapid, non-separation electrochemiluminescent DNA hybridization assays for PCR products using 3'-labeled ohgonucleotide probes is described by Gudibande, et a/ , Molecular and Cellular Probes, 6 495-503 (1992) A related disclosure is found in international patent application WO 95/08644 A1) Marmaro, et al .(Meeting of the American Association of Clinical Chemists, San
Diego, California November 1994 Poster No 54) discusses the design and use of fluorogenic probes in TaqMan, a homogeneous PCR assay
A PCR-based assay that utilizes the inherent 5' nuclease of rTth DNA polymerase for the quantitative detection of HCV RNA is disclosed by Tsang, et al , (94th General Meeting of the American Society for Microbiology, Las Vegas NE 5/94,
Poster No C376)
Kemp, et al , Gene, 94 223-228 (1990), discloses simplified coloπmetπc analysis of polymerase chain reactions and detection of HIV sequences in AIDS patients
German patent application DE 4234086-A1 (92 02 05) (Henco, et al ) discusses the determination of nucleic acid sequences amplified in vitro in enclosed reaction zone where probe(s) capable of interacting with target sequence is present during or after amplification and spectroscopically measurable parameters of probe undergo change thereby generating signal
U S Patent No 5,232,829 (Longiaru, et al ) discloses detection of chlamydia trachomatis by polymerase chain reaction using biotin labeled DNA primers and capture probes A similar disclosure is made by Loeffelholz, et al Journal of Clinical Microbiology, 30(1 1) 2847-2851 (1992)
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
5,679,512
Formation of a single base mismatch that impedes spontaneous DNA branch migration is described by Panyutin, et al (1993) J Mol Biol , 230 413-424
The kinetics of spontaneous DNA branch migration is discussed by Panyutin, et a/ , (1994) Proc Natl Acad Sci USA 91 2021-2025
European Patent Application No 0 450 370 A1 (Wetmur, et al ) discloses branch migration of nucleotides
A displacement polynucleotide assay method and polynucleotide complex reagent therefor is discussed in U S Patent No 4, 766,062 (Diamond, et al ) A strand displacement assay and complex useful therefor is discussed in PCT application WO 94/06937 (Eadie, et al)
PCT application WO/86/06412 (Fπtsch, et al ) discusses process and nucleic acid construct for producing reagent complexes useful in determining target nucleotide sequences SUMMARY OF THE INVENTION
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 When the template is DNA, the invention provides for a denaturation step
In another aspect of the invention, 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 In this primer scheme, either (1) the first primer has a first label and the second primer has a second label or (2) the third primer is a mixture of the third primer with the first label and the third primer with the second label After amplification, 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 a difference is present strand exchange in the complex ceases The association of the first label and the second label is detected The association of the labels being related to the presence of the difference
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 In one aspect of the invention, 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
BRIEF DESCRIPTION OF THE DRAWINGS 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
DETAILED DESCRIPTION OF THE INVENTION 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 Thus, 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
Before proceeding further with a description of the specific embodiments of the present invention, a number of terms will be defined 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 unpuπfied form including DNA (dsDNA and ssDNA) and RNA, including t-RNA, m-RNA, r-RNA, mitochondria! DNA and RNA, chloroplast DNA and RNA, DNA-RNA hybrids, or mixtures thereof, genes, chromosomes, plasmids, the genomes of biological material such as microorganisms, e g , bacteria, yeasts, viruses, viroids, molds, fungi, plants, animals, humans, cDNA, and the like The 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 transcπptase The nucleotide polymerase used in the present invention for carrying out amplification and chain extension can have reverse transcπptase activity Sequences of interest may be embedded in sequences of any length of the chromosome, cDNA, plasmid, etc Sample — the material suspected of containing the nucleic acid Such samples include biological fluids such as blood, serum, plasma, sputum, lymphatic fluid, semen, vaginal mucus, feces, urine, spinal fluid, and the like, biological tissue such as hair and skin, and so forth Other samples include cell cultures and the like, plants, food, forensic samples such as paper, fabrics and scrapings, water sewage, mediαnals, etc When necessary, the sample may be pretreated with reagents to liquefy the sample and release the nucleic acids from binding substances Such pretreatments are well known in the art Amplification of nucleic acids - any method that results in the formation of one or more copies of a nucleic acid Numerous methods are known including the polymerase chain reaction (PCR), ligase chain reaction (LCR) amplification using Q beta rep case, nucleic acid sequence based amplification (NASBA), single primer amplification (ASPP) and others References to these and other amplification methods are provided in the "Description of the Related Art" section, supra
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 Usually, 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, more frequently, 100 to 10,000 nucleotides, preferably, 50 to 1 ,000 nucleotides The target nucleic acid sequence is generally a fraction of a larger molecule or it may be substantially the entire molecule The minimum number of nucleotides in the target sequence is selected to assure that a determination of a difference between two related nucleic acid sequences in accordance with the present invention can be achieved
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
Consequently, both the reference and the target nucleic acid sequences are subjected to similar or the same amplification conditions As with the target nucleic acid sequence, 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 sequences and their complementary sequences The junction is capable of undergoing branch migration resulting in dissociation into two double stranded sequences where sequence identity and complementarity extend to the ends of the strands Quadromolecular Complex -- a complex of four nucleic acid strands containing a Holhday junction which is inhibited from dissociation into two double stranded sequences because of a difference in the sequences and their complements Related nucleic acid sequences - two nucleic acid sequences are related when they contain at least 15 nucleotides at each end that are identical but have different lengths or have intervening sequences that differ by at least one nucleotide Frequently, related nucleic acid sequences differ from each other by a single nucleotide Such difference is referred to herein as the "difference between two related nucleic acid sequences " A difference can be produced by the substitution, deletion or insertion of any single nucleotide or a series of nucleotides within a sequence
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 ohgonucleotιde(s) are usually comprised of a sequence of 10 to 100 nucleotides, preferably, 20 to 80 nucleotides in length
Various techniques can be employed for preparing an ohgonucleotide utilized in the present invention Such ohgonucleotide can be obtained by biological synthesis or by chemical synthesis For short sequences (up to about 100 nucleotides) chemical synthesis will frequently be more economical as compared to the biological synthesis In addition to economy, chemical synthesis provides a convenient way of incorporating low molecular weight compounds and/or modified bases during the synthesis step Furthermore, chemical synthesis is very flexible in the choice of length and region of the target polynucleotide binding sequence The 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 For longer sequences standard replication methods employed in molecular biology can be used such as the use of M13 for single stranded DNA as described by Messing J , Methods Enzymol, 101 20-78 (1983)
Other methods of ohgonucleotide synthesis include phosphotπester 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 pπmer(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, containing a hybπdizable sequence at its 3'-end that is capable of hybridizing with a defined sequence of the target or reference polynucleotide Normally, the hybπdizable sequence of the ohgonucleotide primer has at least 90%, preferably 95%, most preferably 100%, complementarity to a defined sequence or primer binding site The number of nucleotides in the hybπdizable sequence of an ohgonucleotide primer should be such that stringency conditions used to hybridize the ohgonucleotide primer will prevent excessive random non-specific hybridization. Usually, the number of nucleotides in the hybπdizable sequence of the ohgonucleotide primer will be at least ten nucleotides, preferably at least 15 nucleotides and, preferably 20 to 50, nucleotides In addition, 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 tπphosphates -- nudeosides having a 5'-tπphosphate substituent
The nudeosides are pentose sugar derivatives of nitrogenous bases of either puπne or pyπmidine derivation, covalently bonded to the 1 '-carbon of the pentose sugar, which is usually a deoxyribose or a πbose The puππe bases comprise adenιne(A), guanine (G), inosine (I), and derivatives and analogs thereof The pyπmidine bases comprise cytosine (C), thymine (T), uracil (U), and derivatives and analogs thereof
Nucleoside tπphosphates include deoxyπbonucleoside tπphosphates such as the four common tπphosphates dATP, dCTP, dGTP and dTTP and πbonucleoside tπphosphates such as the four common tπphosphates rATP, rCTP, rGTP and rUTP. The term "nucleoside tπphosphates" 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 tπphosphates Examples of such derivatives or analogs, by way of illustration and not limitation, are those which are biotinylated, amine modified, alkylated, and the like and also include phosphorothioate, phosphite, ring atom modified derivatives, and the like
Nucleotide -- a base-sugar-phosphate combination that is the monomenc unit of nucleic acid polymers, i e , DNA and RNA
Nucleoside - is a base-sugar combination or a nucleotide lacking a phosphate moiety
Nucleotide polymerase -- a catalyst, usually an enzyme, for forming an extension of a polynucleotide along a DNA or RNA template where the extension is complementary thereto The nucleotide polymerase is a template dependent polynucleotide polymerase and utilizes nucleoside tπphosphates as building blocks for extending the 3'-end of a polynucleotide to provide a sequence complementary with the polynucleotide template Usually, the catalysts are enzymes, such as DNA polymerases, for example, prokaryotic DNA polymerase (I, II, or III), T4 DNA polymerase, T7 DNA polymerase, Klenow fragment, and reverse transcπptase, 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, animals, virus, thermophihc bacteria, and so forth
Wholly or partially sequentially -- when the sample and various agents utilized in the present invention are combined other than concomitantly (simultaneously), one or more may be combined with one or more of the remaining agents to form a subcombination Subcombination and remaining agents can then be combined and can be subjected to the present method
Hybridization (hybridizing) and binding - in the context of nucleotide sequences these terms are used interchangeably herein The ability of two nucleotide sequences to hybridize with each other is based on the degree of complementarity of the two nucleotide sequences which in turn is based on the fraction of matched complementary nucleotide pairs The more nucleotides in a given sequence that are complementary to another sequence, the more stringent the conditions can be for hybπdization and the more specific will be the binding of the two sequences Increased stringency is achieved by elevating the temperature, increasing the ratio of cosolvents, lowering the salt concentration, and the like
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 hybπzing 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 tπphosphates 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
Member of a specific binding pair ("sbp member") -- one of two different molecules, having an area on the surface or in a cavity which specifically binds to and is thereby defined as complementary with a particular spatial and polar organization of the other molecule The members of the specific binding pair are referred to as hgand and receptor (antihgand) These may be 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 ("antihgand") -- 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
1500, preferably 100 to 1000, more preferably 300 to 600 such as biotin, digoxin, fluorescein, rhodamine and other dyes, tetracycline and other protein binding molecules, and haptens, etc. The small organic molecule can provide a means for at- tachment of a nucleotide sequence to a label or to a support.
Support or surface -- a porous or non-porous water insoluble material. The support can be hydrophilic or capable of being rendered hydrophilic and includes inorganic powders such as silica, magnesium sulfate, and alumina; natural polymeric materials, particularly cellulosic materials and materials derived from cellulose, such as fiber containing papers, e.g., filter paper, chromatographic paper, etc.; synthetic or modified naturally occurring polymers, such as nitrocellulose, cellulose acetate, poly (vinyl chloride), polyacrylamide, cross linked dextran, agarose, polyacrylate, polyethylene, polypropylene, poly(4-methylbutene), polystyrene, 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 be employed.
Binding of sbp members to a support or surface may be accomplished by well- known techniques, commonly available in the literature. See, for example, Immobilized Enzymes, Ichiro Chibata, Halsted Press, New York (1978) and Cuatrecasas,
J.Biol.Chem., 245:3059 (1970). The surface can have any one of a number of shapes, such as strip, rod, particle, including bead, and the like.
Label - a member of a signal producing system. 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. As mentioned above, a reporter molecule can serve as a label and can be bound directly to a nucleotide sequence. Alternatively, 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.
Signal Producing System — 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. When a reporter molecule is not conjugated to a nucleotide sequence, 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. Other components of 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.
Ancillary Materials -- Various ancillary materials will frequently be employed in the methods and assays carried out in accordance with the present invention. For example, buffers will normally be present in the assay medium, as well as stabilizers for the assay medium and the assay components. Frequently, in addition to these additives, proteins may be included, such as albumins, organic solvents such as formamide, quaternary ammonium salts, polycations such as dextran sulfate, surfactants, particularly non-ionic surfactants, binding enhancers, e g , polyalkylene glycols, or the like
Template Switch Isothermal Amplification
In general, 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
Referring now to FIG 1 a, 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'-taιl portion This 5'-portιon 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 design parameters for this section of the TSO, A' and A are described in detail in Patel R, et al, Formation of chimeπc DNA primer extension by template switching onto an annealed downstream ohgonucleotide, PNAS, 93.2969-74 (1996) and U.S Patent No 5,679,512 Briefly, the efficiency of template switching is influenced by the sequence element of the TSO and by reaction conditions The switch of primer extension from the original template to the unhybπdized portion of the TSO results from initial primer extension into the double stranded portion at the site of hybridization of the TSO to the target strand, with concomitant displacement of a portion of the TSO As primer extension proceeds through the double stranded region, a competition is set up between the 3' end of the extending strand and the segment of the displaced TSO for hybridization to the target template, which reaches an equilibrium states. 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.
The hybridization of the primer extension product to the TSO segment 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. In order to achieve efficient extension into the double stranded segment and subsequent template switching, 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. Referring now to FIG. 1b, 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
P2 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 transcπptase, 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 transcπptase 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 (or the temperature suitable for activity of the enzymes used for target amplification) Dependent on the Tm of the target complementary sequences of the TSO and P1 , these oligonucleotides will hybridize to the target at either 65°C or 41 °C, to form complex I (FIG 1a) P1 hybridizes to the target at a position which is 3' to the TSO hybridization sequence Following a short incubation at 41 °C, the amplification enzymes are added Referring to FIG 1a, the reverse transcπptase 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'- portion of TSO (which is not complementary to the target) This process results in the production of three-stranded structure complex II, which includes a double-stranded
DNA portion forming the promoter of the DNA-dependent RNA polymerase The RNase H enzyme activity then degrades the RNA target in the portions of the RNA/DNA hybrid to form complex III
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 Thus, a cycle for exponential amplification of the initial target nucleic acid is established
Referring now to FIG 1 b, 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 Pπmer 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 In addition, 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 In the currently known transcription-based amplification methods, target amplification follows the hybridization of a single primer which is 5'-taιled 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
The discrimination of an RNA gene sequence from a DNA 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 In the present isothermal template-switch amplification, 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 Thus, it is highly unlikely that amplification of a DNA sequence could proceed without full denaturation of the double-stranded DNA target
Refeπng now to FIG 2, 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) Depending on the Tm of the target complementary sequences of the TSO and P1 , 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
As in the case of amplification of an RNA target, the reverse transcπptase 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'-portιon 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 transcπptase 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
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 As depicted in FIG 1 b, 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 product, as described previously herein
It should be noted that 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
It should also be noted, that the 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 Also, the restrictive requirement of formation of a tπ-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
For specific amplification of a DNA target sequence in the presence of RNA targets, usually mRNA, it is desirable to design the amplification primers and TSO to be complementary to sequences of the DNA target strand which are not included in the mRNA Alternatively, 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
The formation of single-stranded 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
Branch Migration Inhibition-based procedure for scanning nucleic acid sequences using the strand-switch isothermal amplification
The Branch Migration Inhibition (BMI) method for the detection of sequence alterations in nucleic acid sequences has been described in WO 97/23646, which is incorporated fully herein by reference The 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 BMI 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 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 In cases where sequence alterations in the expressed gene region are suspected, it may be desirable to use mRNA as initial target molecules, as these are present in excess relative to the genomic sequence As in any amplification-based method for assessing sequence alteration, it is desirable to limit the extent of in vitro amplification, due to potential replication errors introduced by the polymerase used, or the less than perfect fidelity of in vitro amplification 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 In cases requiring analysis of genomic sequences, 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 duplexes which in turn anneal to form four-stranded cruciform structures When the four-stranded cruciform structures are formed in a mixture of test and de¬
reference amplification products, strand exchange by spontaneous branch migration proceeds if the test and reference amplification products are identical When the test sequence is different from the reference branch migration is inhibited, resulting in the formation of stable, detectable four stranded cruciform structures The utility of branch migration inhibition in the detection of sequence alteration in a test sequence relative to a reference sequence was made possible by the invention of a PCR scheme using specially designed primers
Referring now to FIG 3, and as explained more fully in WO 97/23646, in one form of the PCR-based BMI method for detection of sequence alteration, 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'-portιon, Pa, which is complementary to the target, and 5'-taιl sequences, A1 and B1 , which are different for the two reverse primers and are not related to the target Following amplification of the test sequence and the reference sequence, 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 primers P1 and P2 shown in FIGS 1 and 2 and as previously described herein Referring now to FIG 4a, primer P1 is replaced by two primers, F1 and F2 Each of these primers has a common 3'-portιon, P1 , which is complementary to the target, and a 5'-taιl, t1 and t2, respectively The sequences t1 and t2 are different from each other and are not related to the target In addition, two P2 primers are used, each having the same sequence as described earlier, but are 5'-labeled with two different labels Biotin and digoxigenin are suiable labels and as described herein more fully, a number of labels are appropriate Alternatively, the two labels may be attached to the 5'-end of F1 and F2 When F1 and F2 are labeled only one P2 primer is used The TSO ohgonucleotide is designed similarly to that employed in amplification of either DNA or RNA target, as described above
The formation of double-stranded DNA products which are capable of formation of the required partial duplexes is described in Figure 4a Amplification of either DNA or RNA target sequences proceeds as described above, using substantially equimolar mixtures of the primers F1and F2 and bιotιn-P2 and Dig -P2, as well as the TSO It is also possible to separate the two 5'-labeled P2 primers so that amplification of the test sequence is carried out with P2 primer labeled with one label and the amplification of the reference sequence with the other labeled P2 primer
As shown in FIG 4a, amplification of both the test and reference sequences is initiated by combining the sample with a reaction mixture composed of the primers, the TSO, NTPs, and a suitable buffer 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 When the target nucleic acid sequence is an RNA molecule, 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 tπ molecular complexes XII (test or reference, TSO and F1) and XIII (test or reference, TSO, and
F2) 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
Referring now to FIG 4b, primer extension by RT 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
Extension of the primer and the single stranded DNA product by RT then follows to produce fully double-stranded DNA products which contain a promoter site for the RNA polymerase at one end, and the double-stranded tails, t1/t1' and t2/L2' at the other end The double stranded products are labeled with either one of the two labels used (e g , biotin and digoxigenin), biotin-or dig-labeled XXIV and biotin- or dig -labeled XXV The 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
When the target sequence is an 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
RNase H 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
At the end of the isothermal amplification of the test and reference target sequences, ahquots of the respective amplification reactions are mixed The RNA products are degraded by RNase and the mixture is heated to 95°C, to allow denaturation of the double-stranded DNA amplification products The mixture is then incubated at a lower temperature, 65°C, to allow annealing of the single strands to form complete and partial duplexes As seen in FIG 5, 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 Thus, the partial duplex is said to be "tailed" because each strand of the duplex has a single stranded ohgonucleotide chain linked thereto
The degradation of the RNA products prior to BMI analysis is required since these products can compete with the DNA products, as follows First, the excess RNA product can anneal to the complementary DNA strands, following denaturation of the double-stranded DNA products, thus interfering with formation of partial DNA duplexes 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 Referπng now to 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 When the test sequence is identical to the reference sequence, branch migration results in strand exchange, formation of fully double-stranded DNA duplexes, and dissociation of the two labels
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, indicating sequence alteration in the test sequence relative to a reference sequence, can be carried out using a variety of methods suitable for the detection of the association of the two labels When the two labels are biotin and digoxigenin, 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 28th Annual
Oakπdge Conference on Advanced Analytical Concepts for the Clinical Laboratory, "Tomorrow's Technology Today", p 15 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
Performance of BMI/LOCI detection of sequence alteration is shown in WO 97/23646
A potential advantage of the new procedure for detection of sequence alteration in a test sequence as compared to a reference sequence, in contrast to the PCR-based method, is the relative resistance of the new amplification method to nonspecific priming Nonspecific PCR priming was shown to lead to formation of stable cruciform structures which are independent of sequence alteration Unlike the PCR based method, mispπming in the current amplification method cannot lead to the formation of amplification products, in so far as products of mispπming 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
In carrying out the present method, 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 In general for amplification, 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,
Tπs, barbital and the like The particular buffer employed is not critical to this invention but in individual methods one buffer may be preferred over another The buffer employed in the present methods normally contains magnesium ion (Mg2+), which is commonly used with many known polymerases, although other metal ions such as manganese have also been used Preferably, 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 Preferably, the concentration of the ohgonucleotide pπmer(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 The concentration of the nucleoside tπphosphates in the medium can vary widely, preferably, these reagents are present in an excess amount for both amplification and chain extension The nucleoside tπphosphates are usually present in 10-6 to 10-2M, preferably 10-5 to 10-3M As mentioned above, the identity of the target nucleic acid sequence does not need to be known except to the extent to allow preparation of the necessary pπmers and TSO for carrying out the above reactions The present invention permits the determination of the presence or absence of a mutation in a nucleic acid sequence in a sample without the need to fully identify the sequence of the nucleic acid Accordingly, one is able to determine the presence of a mutation in a nucleic acid sequence that is flanked by two sequences of nucleotides for which primers can be made
Detection
We have described a process for the detection of any sequence alteration in a target molecule relative to a reference sequence, based on amplification and BMI analysis of the amplified sequences 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 Alternatively, 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 detection of two different entities is pertinent to the present invention
(1) The detection of the single stranded RNA products generated by the isothermal amplification, and (2) The detection of the stable cruciform complex generated by the BMI analysis of test and reference nucleic acid sequence, if a difference is present between the related sequences
One means of detecting the RNA product generated by the amplification of the present invention is formation of a tπmolecular 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 tπmolecular 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
Similarly, in the present invention 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 Generally, 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 tπmolecular complex or the quadramolecular complexes of the present invention The association of the labels within the complex may be detected in many ways
For example, 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 An example of a system as described above is the enzyme-linked immunosorbent assay (ELISA) a description of which is found in Enzyme-lmmunoassay Edward T Maggio, editor, CRC Press Inc , Boca Raton Florida (1980) wherein for example, the sbp member is biotin, the complementary sbp member is streptavidin and 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 signal producing system would include enzyme substrates and so forth The product of the enzyme reaction is preferably a luminescent product, or a fluorescent or non- fluorescent dye, any of which can be detected spectrophotometπcally, or a product that can be detected by other spectrometπc or electrometπc means If the reporter molecule is a fluorescent molecule, the medium can be irradiated and the fluorescence determined Where the label is a radioactive group, the medium can be counted to determine the radioactive count
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 In one aspect of the present invention detection of the complex is accomplished by employing at least one suspendable particle as a support, which may be bound directly to a nucleic acid strand or may be bound to an sbp member that is complementary to an sbp member attached to a nucleic acid strand 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 labels that may be used in this particular embodiment are fluorescent labels, particles containing a sensitizer and a chemiluminescent olefin (see U S Patent No 5,709,994, the disclosure of which is incorporated herein by reference), chemiluminescent and electroluminescent labels
Preferably, the particle itself can serve as part of a signal producing system that can function without separation or segregation The second label is also part of the signal producing system and can produce a signal in concert with the particle to provide a homogeneous assay detection method A variety of combinations of labels can be used for this purpose When all the reagents are added at the beginning of the reaction, the labels are limited to those that are stable to the elevated temperatures used for amplification, chain extension and branch migration In that regard it is desirable to employ as labels 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 polyπbonucleotides, polynucleoside phosphonates, peptido-nucleic acids, polynucleoside phosphorothioates, homo DNA
In general uncharged nucleic acid analogs provide stronger binding and shorter sequences can be used Included in the reaction medium are 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 such as, for example, carbon, hydrogen, nitrogen, oxygen, sulfur, and phosphorus, which may be present as various groups such as polymethylenes, polymethylene ethers hydroxylated polymethylenes, and so forth The abasic group conveniently may be introduced into the primer during solid phase synthesis by standard methods
Under the proper annealing temperature 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 tπmolecular 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
When 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 When detection of the single stranded product is carried out using two labeled probes, a tπmolecular complex is formed, and the two polynucleotide analog or ohgonucleotide labels, each attached to the corresponding probe, become associated within the complex Alternatively, the single stranded amplification product can be detected by using one labeled probe and one labeled primer, each comprising a polynucleotide analog or an ohgonucleotide label as described above
In the present invention 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 Referring to Figure 7, 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', which is complementary to the label sequence L2, is attached to the second signal generating particle, which may be a sensitizer The probes and signal generating particles may be added to the reaction mixture following amplification or may be included in the amplification reaction mixture The ohgonucleotide probes hybridize to the amplification product Probe 1 bind to the amplification product by hybridization of sequence PS1 to sequence TS1 Probe 2 similarly binds to the amplification product by hybridization of sequence PS2 to sequence TS2 The tπmolecular complex thus formed comprises the single stranded RNA product and the labeled oligonucleotides probes Binding of the signal generating particles to the tπmolecular complex proceeds as described in the following
Under proper annealing temperature 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 As mentioned above, by using different ohgonucleotide or polynucleotide analog sequences for labels, two different reporter groups or particles can become part of the complex Various combinations of particles and reporter groups can be used The tπmolecular complexes or the quadramolecular complexes can also be detected using other hgand/receptor combinations For example, the two labels attached to the primers or probes may be small molecules such as biotin and digoxigenin and the two corresponding receptors can be streptavidin and anti digoxin antibody The attachment of the labels to the ohgonucleotide primers or probes is carried out by methods known in the art The receptors are attached to the reporter molecule or particles Under proper conditions the receptors for example streptavidin and anti digoxin antibody, attached to a reporter molecule or a particle, bind to the corresponding labels, biotin and digoxin respectively, attached to the primers or probes If the labels become part of the complex, the reporter molecule or particle becomes part of the complex The association of the reporter groups or particles is detected Various combinations of particles and reporter groups can be used
The particles, for example, 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 tπtiated 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 U S Patent No 4,868,104
Briefly, 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 By way of illustration as applied to the present invention, 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
These particles have a recognition sequence attached thereto, which is different than that attached to the chemiluminescent particles Once the medium has been treated in accordance with the present invention to form a complex, either the tπmolecular complex or a quadramolecular complex, the medium is irradiated with light to excite the photosensitizer, which is capable in its excited state of activating oxygen to a singlet state Because 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 As a matter of convenience predetermined amounts of reagents employed in the present invention can be provided in a kit in packaged combination A kit can comprise in packaged combination (a) a template switch ohgonucleotide, a first primer, and a second primer as described herein The kit can also include a reference nucleic acid, which corresponds to a target nucleic acid sequence except for the possible presence of a difference such as a mutation, a third and fourth primer, and reagents for conducting an amplification of target nucleic acid sequence prior to subjecting the target nucleic acid sequence to the methods of the present invention The kit can also include nucleoside tπphosphates and a nucleotide polymerase The kit can further include particles as described above capable of binding to the label on at least one of the primers or probes The kit can further include members of a signal producing system and also various buffered media some of which may contain one or more of the above reagents Preferably, at least all of the above components other than buffer are packaged in a single container The relative amounts of the various reagents in the kits can be varied widely to provide for concentrations of the reagents which substantially optimize the reactions that need to occur during the present method and to further substantially optimize the sensitivity of the method in detecting a mutation Under appropriate circumstances one or more of the reagents in the kit can be provided as a dry powder, usually lyophi zed, including excipients, which on dissolution will provide for a reagent solution having the appropriate concentrations for performing a method or assay in accordance with the present invention Each reagent can be packaged in separate containers or some or all of the reagents can be combined in one container where cross-reactivity and shelf life permit In a particular embodiment of a kit in accordance with the present invention, the reagents are packaged in a single container The kits may also include a written description of a method in accordance with the present invention as described above
EXAMPLES Materials:
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
O go Etc (Wilsonville OR) and Operon Technology Inc (Alameda, CA)
Melting temperatures and priming efficiency for individual oligonucleotides were determined by using the OLIGO computer analysis program Molecular weight size markers (50 to 2kb) were purchased from Bio-Rad (Hercules,
CA)
Cloned Pfu polymeras was purchased from Stratagene (LaJolla, CA)
Reverse Transcπptase, T7 RNA polymerase, RNase H and an RNase A inhibitor "RNA guard" were purchased from Pharmacia Biotech (Piscataway, NJ) Ultrapure nucleoside 5'-trιphosphate (rNTP) and 2'deoxynucleosιde 5'-tπphosphates
(dNTPs) were purchased as 100 mM solution from Pharmacia Biotech (Piscataway,
NJ)
Bovine Serum Albumin (BSA) 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)
4-10% precast polyacrylamide (native) gels were purchased from Novex (San Diego,
CA) and used to analyze the amp cation products
Buffer A 10mM Tπs-HCI, pH 8 3, 50mM KCl, 1 5mM MgCI2, and 200μg/ml BSA (used for PCR amplification reactions) Buffer B 40mM Tπs-HCI, pH 8 5, 5mM DTT, 12mM MgCI2, 70mM KCl, 108 8 μg/ml
BSA
Buffer C 50 mM KCl , 4 mM MgCI2, 10 mM Tris-HCl pH 8 3 , 200 μg/ml BSA (used for
LOCI bead suspension)
Abbreviations:
PCR Polymerase Chain Reaction
NASA Nucleic Acid Sequence Amplification Analysis dNTPs 2'-deoxynucleosιde 5'-tπphosphates cloned Pfu Cloned Pyrococus fuπosus DNA polymerase RT Reverse Transcπptase
RNase H Cloned Ribonuclease H
RNA guard RNase A Inhibitor from Human Placenta
TSO Template Switching Ohgonucleotide BSA Bovine Serum Albumin
LOCI Luminescent Oxygen Channeling Immunoassay
DMSO Dimethyl Sulfoxide
Target Nucleic Acid Sequences
1 E coh DNA J gene (1358 bp), complete cds GenBank Accession number M12565
2 M tuberculosis rpoB gene (3853 bp), partial cds GenBank Accession number U12205
OLIGONUCLEOTIDE SEQUENCES
Underlined sequences denote sequences complementary to target or product sequences E coh DNA J gene
TSO 5' - AAT TCT AAT ACG ACT CAC TAT AGG GAG AGA TCG AGT AGC TCC CTG ACA GTG CAC TGT CAG G GC CGC GGT ACG CTG ATC
AAA GAT CC - 3' (Seq ID No 1) Primer 1 5'-CTG TAC GTG GCG TGA CCA AAG AGA - 3' (Seq ID No 2)]
Primer 2 5'-AAT TCT AAT ACG ACT CAC TAT AGG G AG AGA TCG AGT AGC
TC-3' (Seq ID No 3) Probe 1 5' - TTT TTT TTT TTT TTT TTT TT A ACC AGG TAC ACA GCC GCA
GAC TT X - 3' (SEQ ID NO 4) (X = C-7 Ammo modified) Probe 2 5' - GTC CGA CCT GTC ATG GTT CTG GT T ACT TAC TTA CTT ACT
TAC T X - 3' (Seq ID No 5) (X = C-7 Ammo modified)
M tuberculosis rpoB gene
TSO 5' - AAT TCT AAT ACG ACT CAC TAT AGG GAG TTT TCC CAG TCA
CAG CGA GCT GAT ATC AGC TCG CC G ACC GTA CGC AGG CGG CGG TTG CCG - 3' (Seq ID No 6) Primer 1 5' - GAT CGG GCA CAT CCG GCC GT - 3' (Seq ID No 7) Primer 2 5' - AAT TCT AAT ACG ACT CAC TAT AGG G AG TTT TCC CAG TCA
CA - 3' (Seq ID No 8) Probe 1 5' - TGA ATT GGC TCA GCT GGC TGG TTA CTT ACT TAC TTA CTT
ACT X - 3' (Seq ID No 9) (X = C-7 Ammo modified) Probe 2 5 - TTT TTT TTT TTT TTT TTT TTG ACA GCG GGT TGT TCT GGT
CCA X - 3 (Seq ID No 10) (X = C-7 Ammo modified)
PREPARATION OF LOCI PARTICLES C-28 Thioxene
To a solution of 4-bromoanιhne (30g, 174mmol) in dry DMF (200mL) was added 1-bromotetradecane (89 3mL, 366mmol) and N,N-dιιsopropylethylamιne (62 2mL, 357mmol) The reaction solution was heated at 90°C for 16 hr under argon before being cooled to room temperature To this reaction solution was again added 1- bromotetradecane (45mL 184mmol) and N,N-dιιsopropylethylamιne (31 mL, 178mmol) and the reaction mixture was heated at 90°C for another 15 hr After cooling, the reaction solution was concentrated inyacuo and the residue was diluted with CH2CI2 (400mL) The CH2CI2 solution was washed with 1 N aqueous NaOH (2x), H2O, and brine, was dried over Na2SO4 and was concentrated in vacuo to yield a dark brown oil (about 110g) Preparative column chromatography on silica gel by a Waters 500 Prep
LC system elutmg with hexane afforded a yellow oil that contained mainly the product (4-bromo-N,N-dι-(tetradeca)-anιhne) along with a minor component 1- bromotetradecane The latter compound was removed from the mixture by vacuum distillation (bp 105-110°C, 0 6mm) to leave 50 2g (51 %) of the product as a brown oil To a mixture of magnesium turnings (9 60g, 395mmol) in dry THF (30mL) under argon was added dropwise a solution of the above substituted aniline product (44 7g, 79mmol) in THF (250mL) A few crystals of iodine were added to initiate the formation of the Gπgnard reagent When the reaction mixture became warm and began to reflux, the addition rate was regulated to maintain a gentle reflux After addition was complete, the mixture was heated at reflux for an additional hour The cooled supernatant solution was transferred via cannula to an addition funnel and added dropwise (over 2 5 hr) to a solution of anhydrous phenylglyoxal (11 7g, 87mmol) in THF (300mL) at -30°C under argon The reaction mixture was gradually warmed to 0°C over 1 hr and stirred for another 30 mm The resulting mixture was poured into a mixture of ice water (800mL) and ethyl acetate (250mL) The organic phase was separated and the aqueous phase was extracted with ethyl acetate (3x) The combined organic phases were washed with H2O (2x), brine and was dried over MgSO4 Evaporation of the solvent gave 48 8g of the crude product as a dark green oily liquid Flash column chromatography of this liquid (gradient elution with hexane, 1 5 98 5, 3 97, 5 95 ethyl acetate hexane) afforded 24 7g (50%) of the benzoin product (LSIMS (C42H69N02) [M-H]+ 618 6, 1H NMR (250 MHz, CDCI3) was consistent with the expected benzoin product To a solution of the benzoin product from above (24 7g, 40mmol) in dry toluene (500mL) was added sequentially 2-mercaptoethanol (25g,
320mmol) and TMSCI (100mL, 788mmol) The reaction solution was heated at reflux for 23 hr under argon before being cooled to room temperature To this was added additional TMSCI (50mL, 394mmol), and the reaction solution was heated at reflux for another 3 hr The resulting solution was cooled, was made basic with cold 2 5N aqueous NaOH and was extracted with CH2CI2 (3x) The combined organic layers were washed with saturated aqueous NaHCO3 (2x) and brine, was dried over Na2SO4 and was concentrated in vacuo to give a brown oily liquid Preparative column chromatography on silica gel by using a Waters 500 Prep LC system (gradient elution with hexane, 1 99, 2 98 ethyl acetate hexane) provided 15 5g (60%) of the C-28 thioxene as an orange-yellow oil (LSIMS (C44H7ιNOS) [M-H]+ 661 6, 1H NMR (250
MHz, CDCI3) was consistent with the expected C-28 thioxene product 2-(4-(N,N-dι- (tetradeca)-anιlιno)-3-phenyl thioxene
Silicon tetra-t-butyl phthalocyanine Sodium metal, freshly cut (5 Og, 208mmol), was added to 300mL of anhydrous methanol in a two-liter, 3-necked flask equipped with a magnetic stirrer, reflux condenser, a drying tube and a gas bubbler After the sodium was completely dissolved, 4-t-butyl-1 ,2-dιcyanobenzene (38 64g, 210mmol, from TCI Chemicals, Portland OR) was added using a funnel The mixture became clear and the temperature increased to about 50°C At this point a continuous stream of anhydrous ammonia gas was introduced through the glass bubbler into the reaction mixture for 1 hr The reaction mixture was then heated under reflux for 4 hr while the stream of ammonia gas continued During the course of the reaction, as solid started to precipitate The resulting suspension was evaporated to dryness (house vacuum) and the residue was suspended in water (400mL) and filtered The solid was dried (60°C, house vacuum, P2O5) The yield of the product (1 ,3-dιιmιnoιsoιndohne, 42 2g) was almost quantitative This material was used for the next step without further purification To a one-liter, three-necked flask equipped with a condenser and a drying tube was added the above product (18g 89mmol) and quinohne (200mL, Aldrich Chemical Company, St Louis MO) Silicon tetrachloπde (11 ml_, 95mmol, Aldrich Chemical Company) was added with a syringe to the stirred solution over a period of 10 minutes After the addition was completed, the reaction mixture was heated to 180- 185°C in an oil bath for 1 hr The reaction was allowed to cool to room temperature and concentrated HCI was carefully added to acidify the reaction mixture (pH 5-6) The dark brown reaction mixture was cooled and filtered The solid was washed with 100mL of water and dried (house vacuum 60°C, P2Os) The solid material was placed in a 1-lιter, round bottom flask and concentrated sulfuπc acid (500mL) was added with stirring The mixture was stirred for 4 hr at 60°C and was then carefully diluted with crushed ice (2000g) The resulting mixture was filtered and the solid wad washed with 100mL of water and dried The dark blue solid was transferred to a 1 -liter, round bottom flask, concentrated ammonia (500mL) was added, and the mixture was heated and stirred under reflux for 2 hr , was cooled to room temperature and was filtered The solid was washed with 50mL of water and dried under vacuum (house vacuum,
60°C, P2O5) to give 12g of product silicon tetra-t-butyl phthalocyan e as a dark blue solid 3-pιcohne (12g, from Aldrich Chemical Company), tπ-n-butyl amine (anhydrous, 40mL) and tπ-n-hexyl chlorosilane (1 1 5g) were added to 12g of the above product in a one-liter, three-necked flask, equipped with a magnetic stirrer and a reflux condenser The mixture was heated under reflux for 1 5 hr and then cooled to room temperature
The picohne was distilled off under high vacuum (oil pump at about 1 mm Hg) to dryness The residue was dissolved in CH2CI2 and purified using a silica gel column (hexane) to give 10g of pure product dι-(tπ-n-hexylsιlyl)-sιlιcon tetra-t-butyl phthalocyanme as a dark blue solid (LSIMS [M-H]+ 1364 2, absorption spectra methanol 674nm (ε 180,000) toluene 678nm,
1H NMR (250 MHz, CDCI3) δ -2 4(m 12H), -1 3(m, 12H), 0 2-0 9 (m, 54H), 1 8(s, 36H), 8 3(d, 4H) and 9 6 (m, 8H) was consistent with the above expected product
Hydroxypropylaminodextran (1 NH2/ 7 glucose) was prepared by dissolving Dextran T-500 (Pharmacia, Uppsala,
Sweden) (50g) in 150 mL of H2O in a 3-neck round-bottom flask equipped with mechanical stirrer and dropping funnel To the above solution was added 18 8g of Zn (BF4)2 and the temperature was brought to 87°C with a hot water bath Epichlorohydπn (350mL) was added dropwise with stirring over about 30 mm while the temperature was maintained at 87-88°C The mixture was stirred for 4 hr while the temperature was maintained between 80°C and 95°C then the mixture was cooled to room temperature Chlorodextran product was precipitated by pouring slowly into 3L of methanol with vigorous stirring, recovered by filtration and dried overnight in a vacuum oven
The chlorodextran product was dissolved in 200mL of water and added to 2L of concentrated aqueous ammonia (36%) This solution was stirred for 4 days at room temperature, then concentrated to about 190mL on a rotary evaporator The concentrate was divided into two equal batches, and each batch was precipitated by pouring slowly into 2L of rapidly stirring methanol The final product was recovered by filtration and dried under vacuum
Hydroxypropylaminodextran (1 NH2/ 7 glucose), prepared above, was dissolved in 50mM MOPS, pH 7 2, at 12 5 mg/mL The solution was stirred for 8 hr at room temperature, stored under refrigeration and centπfuged for 45 m at 15,000 rpm in a Sorvall RC-5B centrifuge immediately before use to remove a trace of solid material
To 10mL of this solution was added 23 1 mg of Sulfo-SMCC in 1 mL of water This mixture was incubated for 1 hr at room temperature and used without further purification Chemiluminescer particles (TAR beads) The following dye composition was employed 20% C-28 thioxene (prepared as described above), 1 6%1-chloro-9,10-bιs(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 by reference The dyeing process involved the addition of the latex beads (10% solids) into a mixture of ethylene glycol (65 4%), 2-ethoxyethanol (32 2%) and 0 1 N NaOH (2 3%) The beads were mixed and heated for 40 minutes at 95°C with continuos stirring While the beads are being heated, the three chemiluminescent dyes were dissolved in 2- ethoxyethanol by heating them to 95°C for 30 minutes with continuous stirring At the end of both incubations, the dye solution was poured into the bead suspension and the resulting mixture was incubated for an additional 20 minutes with continuous stirring Following the 20-mιnute incubation, the beads were removed form the oil bath and are allowed to cool to 40°C ± 10°C The beads were then passed through a 43- micron mesh polyester filter and washed The dyed particles were washed using a Microgon (Microgon Inc , Laguna Hills CA) The beads were first washed with a solvent mixture composed of ethylene glycol and 2-ethoxyethanol (70%/30%) The beads were washed with 500 ml of solvent mixture per gram of beads This is followed by a 10 % aqueous ethanol (pH 10-1 1) wash The wash volume was 400 ml per gram of beads The beads were then collected and tested for % solid, dye content, particle size, signal and background generation
Ohgonucleotide Bound Chemiluminescer Particles
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 H2O) The sulfo-SMCC was added to a solution of the aminodextran (in 40 mL, 0 05 M Na2HPO4, 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 centπfuged at 15,000 rpm for 15 minutes and the supernatant collected The supernatant dextran solution (54 mL) was then treated with imidazole (7 mL of 1 0 M solution) in MES buffer
(pH 6 0) and into this stirred solution was added the stained chemiluminescer particles (10 mL of 10mg/ml_) After stirring for 10 minutes the suspension was treated with EDAC (7 mmol in 10 mM pH 6 0 MES) and the suspension stirred for 30 minutes After this time, SurfactAmps® (Pierce) Tween-20 (10%, 0 780 mL) was added to the reaction mixture for a final concentration of 0 1 % The particles were then centπfuged at 15,000 rpm for 45 minutes and the supernatant discarded The pellet was resuspended in MES/NaCI (pH 6 0, 10 mM, 100 mL) by sonication Centπfugation at 15,000 rpm for 45 minutes, followed by pellet resuspension after discarding the supernatant, was performed twice The maleimidated dextran chemiluminescer particles were stored in water as a 10 mg/mL suspension
Thiolated ohgonucleotide (ohgonucleotide bearing a 5'-bιs(6- hydroxyethyldisulfide) group) (Ohgos Etc ) was dissolved in water at a concentration of 0 49 mM To 116 μL of this solution was added 8 3 μL of 3 5 M sodium acetate, pH 5 3 and 8 9 μL of trιs(carboxyethyl)phosphιne (20 mM) After 30 minutes incubation at room temperature 548 μL of cold ethanol was added and the mixture was maintained at about 20°C for 1 5 hour The precipitated ohgonucleotide was recovered by centπfugation for 2 mm at 15,000 rpm in an Eppendorf centrifuge, then dissolved in 37 5 μL of 5mM sodium phosphate, 2 mM EDTA, pH 6
An aliquot of the maleimidated beads prepared above containing 22 mg beads was centπfuged for 30 m at about 37,000 g, and the pellet was resuspended in 96 μL of 0 26 M NaCI, 0 05% Tween-20 95 mM sodium phosphate, and 0 95 mM EDTA, pH7 The thiolated ohgonucleotide was added and the mixture was maintained at 37°C for 64 hours under argon A 10 μL aliquot of sodium thioglycolate was added and incubation was continued for 2 hours at 37°C Water was added to a total volume of 1 mL, and the beads were recovered by centπfugation, then resuspended in 5 mL of 0 1 M NaCI, 0 17 M glycme, 10 tng/mL BSA, 1 mM EDTA, 0 1 % Tween-20, and 0 5 mg/mL Calf thymus DNA (Sigma Molecular Biology grade), pH 9 2 After three hours, the beads were recovered and washed three times by centπfugation, twice in buffer A and once in standard PCR buffer The product was stored refrigerated in PCR buffer Buffer A contained 0 1 M Tπs base (J T Baker Chemical Co ), 0 3 M NaCI (Malhnckrodt), 25 mM EDTA Na2 H2O (Sigma Chemical Co ), 0 1 % BSA (Sigma Chemical Co ), 0 1 % dextran (Pharmacia), HBR-1 (Scantibodies), 0 05% Kathon and 0 01 % gentamicm sulfate (GIBCO) prepared by dissolving and adjusting pH to 8 20 with concentrated HCI and made up to 10 L with distilled water
Sensitizer Particles
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 60+/-5°C and 85 mL was added to the above round bottom flask through the septum by means of a syringe heated to 120+/-10°C at a rate of 3 mL per min The remaining 85 mL of the phthalocyanme solution was then added as described above The syringe and flask originally containing the phthalocyanme was rinsed with 40 mL of benzyl alcohol and transferred to round-bottom flask After 15 mm 900 mL of deionized water and 75 mL of 0 1 N NaOH was added dropwise over 40 mm The temperature of the oil bath was allowed to drop slowly to 40+/-10°C and stirring was then discontinued The beads were then filtered through a 43 micron polyester filter and subjected to a Microgon tangential flow filtration apparatus (Microgon Inc , Laguna
Hills, CA) using ethanol water, 100 0 to 10 90, and then filtered through a 43 micron polyester filter The beads were then collected and tested for percent solid dye content, particle size, and singlet oxygen generation
Ohgonucleotide Bound Sensitizer Particles
Ohgonucleotide bound sensitizer particles were prepared in a manner similar to that described above for ohgonucleotide bound to chemiluminescer particles
Example 1
Detection of E coh DNAJ target sequence by NASBA/LOCI
A schematic description of formation of detectable complex comprising the RNA amplification product and two labeled probes is shown in FIG 7 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 tπ 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 tπ molecular complex and two signal generating particles for example a chemiluminescer particle and a sensitizer particle as used in LOCI When the L1 and L2 labels are ohgonucleotide re¬
sequences, complementary ohgonucleotide sequences are attached to the respective particles, to enable binding of the two particles to the respective probe label Thus, ohgonucleotide L1 ' which comprises a sequence complementary to L1 is attached to the first particle, and 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'
In a specific example, 13 0 μl 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 μl sample containing E coh genomic DNA was then added to each tube, followed by 15 μl mineral oil
The mixture was incubated at 95°C for 5 mm , cooled down to 41 °C and incubated for 5 mm 5 0 μl 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 Tπs 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 μl 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 μl 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
LOCI signals were obtained using a LOCI strip-reader by irradiation for 1 sec and read for 1 sec for a total of 3 cycles The results are summarized in Table 1 Table 1
Amplification and detection of E co target Using NASBA/LOCI
Figure imgf000050_0001
As shown in Table 1 , 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
Example 2
Improved Amplification of DNA Nucleic acid Target By the Addition Of Pfu DNA Polymerase
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
LOCI detection of NASA amplification products was carried out as in Example 1 The results are summarized in Table 2
Table 2
NASA of E coh DNA gene target with or without Pfu DNA polymerase added at 65°C
Figure imgf000051_0001
As shown in Table 2, a ten fold increase in the sensitivity of amplification and detection of the target nucleic acid is achieved by this modification, as compared to amplification performed without the addition of Pfu to reach a detection limit of about 100 molecules It is likely that Pfu affords a higher efficiency of primer extension and target switch
Example 3 Effect of Incubation Temperature For Pfu Catalyzed Primer Extension and Target
Switch Step
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 effect of incubation temperature for the first primer extension and target switch step, and the efficiency of Pfu polymerase as a separate reagent or as a combined reagent with the three other amplification enzyme, was assessed using two target nucleic acids, the DNAJ gene of E coh and the rpoB gene of M tuberculosis
The results of NASA amplification of these genomic nucleic acid targets and LOCI detection of the amplification products are summarized in Table 3 and Table 4, respectively
Table 3 NASA amplification of E coh DNAJ Target Sequence With or Without Pfu
(as a separate reagent or a combined reagent added at 41 °C)
Figure imgf000052_0001
Table 4
NASA Amplification of M tuberculosis rpoB gene sequence Using TSO1 With or Without Pfu (as a separate reagent or a combined reagent @ 41 °C)
Figure imgf000053_0001
As shown in Tables 3 and 4, 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
The above discussion includes certain theories as to mechanisms involved in the present invention These theories should not be construed to limit the present invention in any way The above description and examples fully disclose the invention including preferred embodiments thereof Modifications of the methods described that are obvious to those of ordinary skill in the art such as molecular biology and related sciences are intended to be within the scope of the claims

Claims

What is claimed is:
1. A method for producing multiple copies of a target polynucleotide sequence comprising: subjecting a mixture of the target polynucleotide sequence, a first ohgonucleotide primer, a template switch oligonucleotide, and reagents sufficient for conducting an amplification of said target polynucleotide sequence, to conditions sufficient for amplifying the target nucleic acid sequence.
2. The method of claim 1 wherein the template switch oligonucleotide comprises a nucleotide sequence having a 3' region capable of hybridizing to the target sequence and a 5' region which does not hybridize to the target sequence.
3. The method according to claim 2 wherein said 5' region of said template switch oligonucleotide includes a propromoter sequence of a DNA dependent RNA polymerase.
4. The method according to claim 3 wherein said 5' region of said template switch oligonucleotide includes a nucleotide sequence substantially homologous to the target sequence located 3' of said propromoter sequence.
5. The method according to claim 4 wherein said 5' region of said template switch oligonucleotide further comprises a sequence unrelated to the target sequence located between said sequence substantially homologous to the target and said propromoter sequence.
6. The method according to claim 5 wherein said sequence homologous to the target sequence is immediately 5' to said 3' region complementary to the target sequence.
7. The method according to claim 1 wherein the conditions for amplifying said target polynucleotide sequence are isothermal.
8. The method according to claim 1 further comprising the addition of a second oligonucleotide primer. 9 The method according to claim 8 wherein said second primer is not target dependant
10 The method according to claim 8 wherein said second primer includes a nucleotide sequence substantially homologous to said sequence unrelated to target sequence on said template switch oligonucleotide
11 The method according to claim 8 wherein said second primer includes the sequence of a propromoter of a DNA dependent RNA polymerase
12 The method according to claim 1 wherein the target is DNA
13 The method according to claim 1 wherein the target is RNA
14 The method according to claim 12 further comprising the step of denaturation of said DNA target
15 The method of claim 1 wherein the reagents sufficient for conducting an amplification include a polynucleotide polymerase deoxynudeoside tπphosphates, and πbonucleoside tπphosphates
16 A method for producing multiple copies of a target nucleic acid sequence comprising hybridizing a first primer and a template switch ohgonucleotide to the target sequence, extending said first primer along the target and along the 5' portion of said template switch oligonucleotide to form a first extension product, transcribing said first extension product to produce multiple copies of a transcription product having a sequence homologous to the target sequence
17 The method of claim 16 further comprising hybridizing said first primer to said transcription product, extending said first primer along said transcription product to form a second extension product, degrading said transcription product, hybridizing a second primer to said second extension product, extending said second primer to form a third extension product, extending said second extension product to produce a double stranded DNA product with said third extension product transcribing said double stranded DNA product to produce multiple copies of a said transcription product
18 The method of claim 16 wherein the template switch ohgonucleotide comprises a nucleotide sequence having a 3' region capable of hybridizing to the target sequence and a 5' region which does not hybridize to the target sequence
19 The method according to claim 18 wherein said 5' region of said template switch o gonucleotide includes a propromoter sequence of a DNA dependent RNA polymerase
20 The method according to claim 19 wherein said 5' region of said template switch o gonucleotide includes a nucleotide sequence substantially homologous to the target sequence located 3' of said propromoter sequence
21 The method according to claim 20 wherein said 5' region of said template switch ohgonucleotide further comprises a sequence unrelated to the target sequence located between said sequence substantially homologous to the target and said propromoter sequence
22 The method according to claim 21 wherein said sequence homologous to the target sequence is immediately 5' to said 3' region complementary to the target sequence
23 The method according to claim 16 wherein the conditions for amplifying said target polynucleotide sequence are isothermal
24 The method according to claim 17 wherein said second primer is not target dependant
25 The method according to claim 17 wherein said second primer includes a nucleotide sequence substantially homologous to said sequence unrelated to target sequence on said template switch ohgonucleotide
26 The method according to claim 17 wherein said second primer includes the sequence of a propromoter of a DNA dependent RNA polymerase 27 The method according to claim 16 wherein the target is DNA
28 The method according to claim 16 wherein the target is RNA
29 The method according to claim 27 further comprising the step of denaturation of said DNA target
30 In an isothermal method for amplifying a target polynucleotide sequence, the improvement comprising using a template switch oligonucleotide and a primer which is not target dependent
31 A method for detecting the presence of a difference between a target nucleic acid sequence and a reference nucleic acid sequence comprising amplifying the target sequence and the reference sequence using a template switch ohgonucleotide, a first primer, a second primer, and a third primer, said first primer and said second primer having common 3' sequences which are complementary to the target and the reference, said first and second primers further having 5' tail sequences which are not complementary to the target sequence, the reference sequence, and each other wherein either
(a) said first primer has a first label and said second primer has a second label, or
(b) said third primer includes a mixture of said third primer having a first label and said third primer having second label, forming a quadramolecular complex including said reference and the target sequences in double stranded form, said complex having at least one pair of non complementary strands and each of the said non complementary strands in the said complex having a label, subjecting the complex to strand exchange conditions, wherein if no difference is present between said test and reference sequences strand exchange continues to completion, and wherein if a difference between said reference and test sequences is present strand exchange in said complex ceases, and detecting the association of said labels within said complex, the association thereof being related to the presence of said difference 32 The method according to claim 31 wherein said first label and said second label are independently selected from the group consisting of oligonucleotides, enzymes, dyes, fluorescent molecules, co-enzymes, enzyme substrates, radioactive groups, small organic molecules, polynucleotide sequences, polynucleotide analog sequences and solid surfaces
33 The method of claim 31 wherein said difference is a mutation
34 The method of claim 31 wherein said complex comprises a Holhday junction
35 The method of claim 31 wherein the amplification of said target sequence and said reference sequence is isothermal
36 The method according to claim 31 wherein said difference is a single nucleotide polymorphism
37 The method according to claim 31 wherein said third primer includes the sequence of a propromoter of a DNA dependent RNA polymerase
38 The method according to claim 31 wherein the target is DNA
39 The method according to claim 31 wherein the target is RNA
40 The method according to claim 38 further comprising the step of denaturation of
41 A method of detecting the presence of a difference between a target nucleic acid sequence and a reference nucleic acid sequence comprising combining the target sequence, a first primer, a second primer, a third primer and a fourth primer, a template switch oligonucleotide, and reagents sufficient to amplify the target sequence, combining in a separate vessel the reference sequence, a first primer, a second primer, a third primer and a fourth primer, a template switch ohgonucleotide, and reagents sufficient to amplify the reference sequence, said first primer and said second primer each having a 3' region which is complementary to the target sequence and the reference sequence and a 5' tail which is not complementary to the target sequence and the reference sequence, said 5' tail of said first primer being non-complementary to said 5' tail of said second primer, said template switch ohgonucleotide having a 3' region which is complementary to the target sequence and the reference sequence, and a 5' region including a sequence unrelated to the target and the reference sequences, said 5' region further including a propromoter of a DNA dependent RNA polymerase said third primer having a first label and said fourth primer having a second label, said third primer and said fourth primer having substantially the same nucleic acid sequences, and said third and fourth primer nucleic acid sequences being substantially homologous to said 5'sequence of the template switch ohgonucleotide which is unrelated to target or reference nucleic acid sequences, forming from said target sequence a tailed target partial duplex of two nucleic acid strands, one of said target strands having said first label or said second label, and each of said strands having non-complementary ohgonucleotide tails, forming from said reference sequence a tailed reference partial duplex of two nucleic acid strands, one of said reference strands having said first label or said second label, and each of said strands having non-complementary oligonucleotide tails forming a quadramolecular complex comprising said tailed target duplex and said tailed reference duplex in double stranded form, wherein said complex includes at least one pair of non-complementary strands and each of said non-complementary strands within said complex has a label, subjecting the complex to strand exchange conditions wherein if no difference is present between the target and the reference sequences, strand exchange continues to completion, and wherein if a difference between said reference and test sequences is present, strand exchange in said complex ceases, and detecting the association of said labels within said complex, the association thereof being related to the presence of the difference
42 The method according to claim 41 wherein said first label and said second label are independently selected from the group consisting of oligonucleotides, enzymes, dyes, fluorescent molecules, co-enzymes, enzyme substrates, radioactive groups, small organic molecules, polynucleotide sequences, polynucleotide analog sequences, and solid surfaces 43 The method of claim 41 wherein said difference is a mutation
44 The method of claim 41 wherein said target sequence is RNA or DNA
45 The method of claim 41 wherein said difference is a single nucleotide polymorphism
46 The method of claim 41 wherein said complex comprises a Holhday junction
47 The method of claims 41 wherein the target sequence, the reference sequences, said primers, said template switch ohgonucleotide and said reagents are combined in the same reaction vessel
48 A kit for producing multiple copies of a target nucleic acid, said kit comprising in packaged form
(a) a template switch ohgonucleotide having the sequence of a propromoter of a DNA dependent RNA polymerase,
(b) a first primer extendable along said target, and said template switch ohgonucleotide
49 The kit according to claim 48 further comprising a second primer have the sequence of a propromoter of a DNA polymerase and a sequence to a portion of the template switch ohgonucleotide which is substantially adjacent to said propromoter sequence on said template switch oligonucleotide
50 The kit according to claim 48 further comprising a first probe a second probe, a first signal generating particle and a second signal generating particle
51 A kit for detecting the presence of a difference between a reference nucleic acid sequence and a target nucleic acid sequence comprising
(a) a reference nucleic acid sequence,
(b) a template switch ohgonucleotide having a sequence of a propromoter of an RNA polymerase,
(c) a first primer and a second primer each comprising a common 3'-end sequence which is complementary to said target and reference nucleic acid sequence, and a 5'-end sequence which is not complementary to said target or reference nuclei acid sequence or to each other, extendable along said target, said reference and said template switch oligonucleotide, (d) a third primer having the sequence of a propromoter of the RNA polymerase and a sequence homologous to a portion of the template switch oligonucleotide which is substantially adjacent to said propromoter sequence on said template switch ohgonucleotide; wherein either said first primer has a first label and said second primer has a second label, or said third primer is a mixture of said third primer with a first label and said third primer with a second label
PCT/US2000/013526 1999-05-17 2000-05-16 Homogeneous isothermal amplification and detection of nucleic acids using a template switch oligonucleotide WO2000070095A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US31324099A 1999-05-17 1999-05-17
US09/313,240 1999-05-17

Publications (2)

Publication Number Publication Date
WO2000070095A2 true WO2000070095A2 (en) 2000-11-23
WO2000070095A3 WO2000070095A3 (en) 2001-08-02

Family

ID=23214934

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2000/013526 WO2000070095A2 (en) 1999-05-17 2000-05-16 Homogeneous isothermal amplification and detection of nucleic acids using a template switch oligonucleotide

Country Status (1)

Country Link
WO (1) WO2000070095A2 (en)

Cited By (53)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001020035A2 (en) * 1999-09-13 2001-03-22 Nugen Technologies, Inc. Methods and compositions for linear isothermal amplification of polynucleotide sequences
WO2002000938A2 (en) * 2000-06-26 2002-01-03 Nugen Technologies, Inc. Methods and compositions for transcription-based nucleic acid amplification
US6692918B2 (en) 1999-09-13 2004-02-17 Nugen Technologies, Inc. Methods and compositions for linear isothermal amplification of polynucleotide sequences
EP1390537A2 (en) * 2001-03-09 2004-02-25 NuGEN Technologies, Inc. Methods and compositions for amplification of rna sequences
JP2007300921A (en) * 2006-05-05 2007-11-22 Qiagen Gmbh Method for inserting sequence element into nucleic acid
US7501254B2 (en) 2006-07-20 2009-03-10 Ghc Technologies, Inc. Methods and compositions for amplification and capture of nucleic acid sequences
US7846733B2 (en) 2000-06-26 2010-12-07 Nugen Technologies, Inc. Methods and compositions for transcription-based nucleic acid amplification
US7846666B2 (en) 2008-03-21 2010-12-07 Nugen Technologies, Inc. Methods of RNA amplification in the presence of DNA
US7939258B2 (en) 2005-09-07 2011-05-10 Nugen Technologies, Inc. Nucleic acid amplification procedure using RNA and DNA composite primers
WO2011091393A1 (en) 2010-01-25 2011-07-28 Rd Biosciences, Inc. Self-folding amplification of target nucleic acid
US8034568B2 (en) 2008-02-12 2011-10-11 Nugen Technologies, Inc. Isothermal nucleic acid amplification methods and compositions
EP2382330A1 (en) * 2009-01-06 2011-11-02 Qimin You Cross priming amplification of target nucleic acids
US8071311B2 (en) 2001-03-09 2011-12-06 Nugen Technologies, Inc. Methods and compositions for amplification of RNA sequences
US8334116B2 (en) 2000-12-13 2012-12-18 Nugen Technologies, Inc. Methods and compositions for generation of multiple copies of nucleic acid sequences and methods of detection thereof
US8465950B2 (en) 2003-04-14 2013-06-18 Nugen Technologies, Inc. Global amplification using a randomly primed composite primer
WO2015200893A3 (en) * 2014-06-26 2016-03-17 10X Genomics, Inc. Methods of analyzing nucleic acids from individual cells or cell populations
US9487828B2 (en) 2012-05-10 2016-11-08 The General Hospital Corporation Methods for determining a nucleotide sequence contiguous to a known target nucleotide sequence
US9644204B2 (en) 2013-02-08 2017-05-09 10X Genomics, Inc. Partitioning and processing of analytes and other species
US9683255B2 (en) 2005-09-09 2017-06-20 Qiagen Gmbh Method for activating a nucleic acid for a polymerase reaction
US9689024B2 (en) 2012-08-14 2017-06-27 10X Genomics, Inc. Methods for droplet-based sample preparation
US9694361B2 (en) 2014-04-10 2017-07-04 10X Genomics, Inc. Fluidic devices, systems, and methods for encapsulating and partitioning reagents, and applications of same
US9701998B2 (en) 2012-12-14 2017-07-11 10X Genomics, Inc. Methods and systems for processing polynucleotides
US9951386B2 (en) 2014-06-26 2018-04-24 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10011872B1 (en) 2016-12-22 2018-07-03 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10221442B2 (en) 2012-08-14 2019-03-05 10X Genomics, Inc. Compositions and methods for sample processing
US10221436B2 (en) 2015-01-12 2019-03-05 10X Genomics, Inc. Processes and systems for preparation of nucleic acid sequencing libraries and libraries prepared using same
US10227648B2 (en) 2012-12-14 2019-03-12 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10273541B2 (en) 2012-08-14 2019-04-30 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10287623B2 (en) 2014-10-29 2019-05-14 10X Genomics, Inc. Methods and compositions for targeted nucleic acid sequencing
CN109790535A (en) * 2016-07-29 2019-05-21 新英格兰生物实验室公司 Prevent the method and composition of concatemerization during template switch
US10323279B2 (en) 2012-08-14 2019-06-18 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10400235B2 (en) 2017-05-26 2019-09-03 10X Genomics, Inc. Single cell analysis of transposase accessible chromatin
US10400280B2 (en) 2012-08-14 2019-09-03 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10428326B2 (en) 2017-01-30 2019-10-01 10X Genomics, Inc. Methods and systems for droplet-based single cell barcoding
US10450597B2 (en) 2014-01-27 2019-10-22 The General Hospital Corporation Methods of preparing nucleic acids for sequencing
US10533221B2 (en) 2012-12-14 2020-01-14 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10550429B2 (en) 2016-12-22 2020-02-04 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10697000B2 (en) 2015-02-24 2020-06-30 10X Genomics, Inc. Partition processing methods and systems
US10745742B2 (en) 2017-11-15 2020-08-18 10X Genomics, Inc. Functionalized gel beads
US10752949B2 (en) 2012-08-14 2020-08-25 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10774370B2 (en) 2015-12-04 2020-09-15 10X Genomics, Inc. Methods and compositions for nucleic acid analysis
US10815525B2 (en) 2016-12-22 2020-10-27 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10829815B2 (en) 2017-11-17 2020-11-10 10X Genomics, Inc. Methods and systems for associating physical and genetic properties of biological particles
US11084036B2 (en) 2016-05-13 2021-08-10 10X Genomics, Inc. Microfluidic systems and methods of use
US11135584B2 (en) 2014-11-05 2021-10-05 10X Genomics, Inc. Instrument systems for integrated sample processing
US11155881B2 (en) 2018-04-06 2021-10-26 10X Genomics, Inc. Systems and methods for quality control in single cell processing
US11241688B2 (en) 2017-06-05 2022-02-08 10X Genomics, Inc. Gaskets for the distribution of pressures in a microfluidic system
US11274343B2 (en) 2015-02-24 2022-03-15 10X Genomics, Inc. Methods and compositions for targeted nucleic acid sequence coverage
US11390905B2 (en) 2016-09-15 2022-07-19 Archerdx, Llc Methods of nucleic acid sample preparation for analysis of DNA
EP3209802B1 (en) * 2014-10-20 2022-09-07 Envirologix Inc. Compositions and methods for detecting an rna virus
US11591637B2 (en) 2012-08-14 2023-02-28 10X Genomics, Inc. Compositions and methods for sample processing
US11773389B2 (en) 2017-05-26 2023-10-03 10X Genomics, Inc. Single cell analysis of transposase accessible chromatin
US11795492B2 (en) 2016-09-15 2023-10-24 ArcherDX, LLC. Methods of nucleic acid sample preparation

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9487823B2 (en) 2002-12-20 2016-11-08 Qiagen Gmbh Nucleic acid amplification

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5545522A (en) * 1989-09-22 1996-08-13 Van Gelder; Russell N. Process for amplifying a target polynucleotide sequence using a single primer-promoter complex
WO1997023646A1 (en) * 1995-12-22 1997-07-03 Behringwerke Aktiengesellschaft Detection of differences in nucleic acids
WO1997024455A2 (en) * 1996-01-03 1997-07-10 Clontech Laboratories, Inc. METHODS AND COMPOSITIONS FOR FULL-LENGTH cDNA CLONING

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5545522A (en) * 1989-09-22 1996-08-13 Van Gelder; Russell N. Process for amplifying a target polynucleotide sequence using a single primer-promoter complex
WO1997023646A1 (en) * 1995-12-22 1997-07-03 Behringwerke Aktiengesellschaft Detection of differences in nucleic acids
WO1997024455A2 (en) * 1996-01-03 1997-07-10 Clontech Laboratories, Inc. METHODS AND COMPOSITIONS FOR FULL-LENGTH cDNA CLONING

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
PANYUTIN I G ET AL: "FORMATION OF A SINGLE BASE MISMATCH IMPEDES SPONTANEOUS DNA BRANCH MIGRATION" JOURNAL OF MOLECULAR BIOLOGY,GB,LONDON, vol. 230, no. 2, 20 March 1993 (1993-03-20), pages 413-424, XP000673372 ISSN: 0022-2836 cited in the application *
PATEL R. ET AL.,: "Formation of chimeric DNA primer extension products by template switching onto an annealed downstream oligonucleotide" PROC. NATL. ACAD. SCI, USA, vol. 93, - April 1996 (1996-04) pages 2969-2974, XP002158856 cited in the application *

Cited By (124)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001020035A2 (en) * 1999-09-13 2001-03-22 Nugen Technologies, Inc. Methods and compositions for linear isothermal amplification of polynucleotide sequences
US6692918B2 (en) 1999-09-13 2004-02-17 Nugen Technologies, Inc. Methods and compositions for linear isothermal amplification of polynucleotide sequences
WO2001020035A3 (en) * 1999-09-13 2001-12-06 Nugen Technologies Inc Methods and compositions for linear isothermal amplification of polynucleotide sequences
WO2002000938A2 (en) * 2000-06-26 2002-01-03 Nugen Technologies, Inc. Methods and compositions for transcription-based nucleic acid amplification
WO2002000938A3 (en) * 2000-06-26 2003-08-28 Nugen Technologies Inc Methods and compositions for transcription-based nucleic acid amplification
US7846733B2 (en) 2000-06-26 2010-12-07 Nugen Technologies, Inc. Methods and compositions for transcription-based nucleic acid amplification
US8334116B2 (en) 2000-12-13 2012-12-18 Nugen Technologies, Inc. Methods and compositions for generation of multiple copies of nucleic acid sequences and methods of detection thereof
US8071311B2 (en) 2001-03-09 2011-12-06 Nugen Technologies, Inc. Methods and compositions for amplification of RNA sequences
EP1390537A4 (en) * 2001-03-09 2006-04-26 Nugen Technologies Inc Methods and compositions for amplification of rna sequences
US9181582B2 (en) 2001-03-09 2015-11-10 Nugen Technologies, Inc. Compositions for amplification of RNA sequences using composite primers
EP1390537A2 (en) * 2001-03-09 2004-02-25 NuGEN Technologies, Inc. Methods and compositions for amplification of rna sequences
US9175325B2 (en) 2003-04-14 2015-11-03 Nugen Technologies, Inc. Global amplification using a randomly primed composite primer
US8465950B2 (en) 2003-04-14 2013-06-18 Nugen Technologies, Inc. Global amplification using a randomly primed composite primer
US8852867B2 (en) 2005-09-07 2014-10-07 Nugen Technologies, Inc. Nucleic acid amplification procedure using RNA and DNA composite primers
US7939258B2 (en) 2005-09-07 2011-05-10 Nugen Technologies, Inc. Nucleic acid amplification procedure using RNA and DNA composite primers
US9683255B2 (en) 2005-09-09 2017-06-20 Qiagen Gmbh Method for activating a nucleic acid for a polymerase reaction
JP2007300921A (en) * 2006-05-05 2007-11-22 Qiagen Gmbh Method for inserting sequence element into nucleic acid
EP1889925A1 (en) 2006-05-05 2008-02-20 Qiagen GmbH Introduction of sequence elements in nucleic acids
EP2316965A1 (en) * 2006-05-05 2011-05-04 Qiagen GmbH Introduction of sequence elements in nucleic acids
US8932831B2 (en) 2006-05-05 2015-01-13 Qiagen Gmbh Insertion of sequence elements into nucleic acids
US7501254B2 (en) 2006-07-20 2009-03-10 Ghc Technologies, Inc. Methods and compositions for amplification and capture of nucleic acid sequences
US8034568B2 (en) 2008-02-12 2011-10-11 Nugen Technologies, Inc. Isothermal nucleic acid amplification methods and compositions
US7846666B2 (en) 2008-03-21 2010-12-07 Nugen Technologies, Inc. Methods of RNA amplification in the presence of DNA
EP2382330A4 (en) * 2009-01-06 2013-02-13 Qimin You Cross priming amplification of target nucleic acids
EP2382330A1 (en) * 2009-01-06 2011-11-02 Qimin You Cross priming amplification of target nucleic acids
US9074246B2 (en) 2010-01-25 2015-07-07 Rd Biosciences, Inc. Self-folding amplification of target nucleic acid
WO2011091393A1 (en) 2010-01-25 2011-07-28 Rd Biosciences, Inc. Self-folding amplification of target nucleic acid
US11781179B2 (en) 2012-05-10 2023-10-10 The General Hospital Corporation Methods for determining a nucleotide sequence contiguous to a known target nucleotide sequence
US9487828B2 (en) 2012-05-10 2016-11-08 The General Hospital Corporation Methods for determining a nucleotide sequence contiguous to a known target nucleotide sequence
US10718009B2 (en) 2012-05-10 2020-07-21 The General Hospital Corporation Methods for determining a nucleotide sequence contiguous to a known target nucleotide sequence
US10017810B2 (en) 2012-05-10 2018-07-10 The General Hospital Corporation Methods for determining a nucleotide sequence contiguous to a known target nucleotide sequence
US11078522B2 (en) 2012-08-14 2021-08-03 10X Genomics, Inc. Capsule array devices and methods of use
US10752950B2 (en) 2012-08-14 2020-08-25 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10597718B2 (en) 2012-08-14 2020-03-24 10X Genomics, Inc. Methods and systems for sample processing polynucleotides
US10626458B2 (en) 2012-08-14 2020-04-21 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10669583B2 (en) 2012-08-14 2020-06-02 10X Genomics, Inc. Method and systems for processing polynucleotides
US10450607B2 (en) 2012-08-14 2019-10-22 10X Genomics, Inc. Methods and systems for processing polynucleotides
US11591637B2 (en) 2012-08-14 2023-02-28 10X Genomics, Inc. Compositions and methods for sample processing
US9689024B2 (en) 2012-08-14 2017-06-27 10X Genomics, Inc. Methods for droplet-based sample preparation
US9695468B2 (en) 2012-08-14 2017-07-04 10X Genomics, Inc. Methods for droplet-based sample preparation
US11441179B2 (en) 2012-08-14 2022-09-13 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10053723B2 (en) 2012-08-14 2018-08-21 10X Genomics, Inc. Capsule array devices and methods of use
US10400280B2 (en) 2012-08-14 2019-09-03 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10584381B2 (en) 2012-08-14 2020-03-10 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10752949B2 (en) 2012-08-14 2020-08-25 10X Genomics, Inc. Methods and systems for processing polynucleotides
US11021749B2 (en) 2012-08-14 2021-06-01 10X Genomics, Inc. Methods and systems for processing polynucleotides
US11035002B2 (en) 2012-08-14 2021-06-15 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10221442B2 (en) 2012-08-14 2019-03-05 10X Genomics, Inc. Compositions and methods for sample processing
US11359239B2 (en) 2012-08-14 2022-06-14 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10323279B2 (en) 2012-08-14 2019-06-18 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10273541B2 (en) 2012-08-14 2019-04-30 10X Genomics, Inc. Methods and systems for processing polynucleotides
US11473138B2 (en) 2012-12-14 2022-10-18 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10612090B2 (en) 2012-12-14 2020-04-07 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10533221B2 (en) 2012-12-14 2020-01-14 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10227648B2 (en) 2012-12-14 2019-03-12 10X Genomics, Inc. Methods and systems for processing polynucleotides
US11421274B2 (en) 2012-12-14 2022-08-23 10X Genomics, Inc. Methods and systems for processing polynucleotides
US9701998B2 (en) 2012-12-14 2017-07-11 10X Genomics, Inc. Methods and systems for processing polynucleotides
US9856530B2 (en) 2012-12-14 2018-01-02 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10676789B2 (en) 2012-12-14 2020-06-09 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10253364B2 (en) 2012-12-14 2019-04-09 10X Genomics, Inc. Method and systems for processing polynucleotides
US10150963B2 (en) 2013-02-08 2018-12-11 10X Genomics, Inc. Partitioning and processing of analytes and other species
US10150964B2 (en) 2013-02-08 2018-12-11 10X Genomics, Inc. Partitioning and processing of analytes and other species
US9644204B2 (en) 2013-02-08 2017-05-09 10X Genomics, Inc. Partitioning and processing of analytes and other species
US11193121B2 (en) 2013-02-08 2021-12-07 10X Genomics, Inc. Partitioning and processing of analytes and other species
US11807897B2 (en) 2014-01-27 2023-11-07 The General Hospital Corporation Methods of preparing nucleic acids for sequencing
US10450597B2 (en) 2014-01-27 2019-10-22 The General Hospital Corporation Methods of preparing nucleic acids for sequencing
US10071377B2 (en) 2014-04-10 2018-09-11 10X Genomics, Inc. Fluidic devices, systems, and methods for encapsulating and partitioning reagents, and applications of same
US10343166B2 (en) 2014-04-10 2019-07-09 10X Genomics, Inc. Fluidic devices, systems, and methods for encapsulating and partitioning reagents, and applications of same
US10150117B2 (en) 2014-04-10 2018-12-11 10X Genomics, Inc. Fluidic devices, systems, and methods for encapsulating and partitioning reagents, and applications of same
US9694361B2 (en) 2014-04-10 2017-07-04 10X Genomics, Inc. Fluidic devices, systems, and methods for encapsulating and partitioning reagents, and applications of same
CN106795553A (en) * 2014-06-26 2017-05-31 10X基因组学有限公司 The method for analyzing the nucleic acid from individual cells or cell colony
US11629344B2 (en) 2014-06-26 2023-04-18 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10208343B2 (en) 2014-06-26 2019-02-19 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10480028B2 (en) 2014-06-26 2019-11-19 10X Genomics, Inc. Methods and systems for processing polynucleotides
US9951386B2 (en) 2014-06-26 2018-04-24 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10457986B2 (en) 2014-06-26 2019-10-29 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10041116B2 (en) 2014-06-26 2018-08-07 10X Genomics, Inc. Methods and systems for processing polynucleotides
CN106795553B (en) * 2014-06-26 2021-06-04 10X基因组学有限公司 Methods of analyzing nucleic acids from individual cells or cell populations
WO2015200893A3 (en) * 2014-06-26 2016-03-17 10X Genomics, Inc. Methods of analyzing nucleic acids from individual cells or cell populations
US10337061B2 (en) 2014-06-26 2019-07-02 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10344329B2 (en) 2014-06-26 2019-07-09 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10760124B2 (en) 2014-06-26 2020-09-01 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10030267B2 (en) 2014-06-26 2018-07-24 10X Genomics, Inc. Methods and systems for processing polynucleotides
US11713457B2 (en) 2014-06-26 2023-08-01 10X Genomics, Inc. Methods and systems for processing polynucleotides
EP3209802B1 (en) * 2014-10-20 2022-09-07 Envirologix Inc. Compositions and methods for detecting an rna virus
US10287623B2 (en) 2014-10-29 2019-05-14 10X Genomics, Inc. Methods and compositions for targeted nucleic acid sequencing
US11739368B2 (en) 2014-10-29 2023-08-29 10X Genomics, Inc. Methods and compositions for targeted nucleic acid sequencing
US11135584B2 (en) 2014-11-05 2021-10-05 10X Genomics, Inc. Instrument systems for integrated sample processing
US10557158B2 (en) 2015-01-12 2020-02-11 10X Genomics, Inc. Processes and systems for preparation of nucleic acid sequencing libraries and libraries prepared using same
US10221436B2 (en) 2015-01-12 2019-03-05 10X Genomics, Inc. Processes and systems for preparation of nucleic acid sequencing libraries and libraries prepared using same
US11414688B2 (en) 2015-01-12 2022-08-16 10X Genomics, Inc. Processes and systems for preparation of nucleic acid sequencing libraries and libraries prepared using same
US10697000B2 (en) 2015-02-24 2020-06-30 10X Genomics, Inc. Partition processing methods and systems
US11603554B2 (en) 2015-02-24 2023-03-14 10X Genomics, Inc. Partition processing methods and systems
US11274343B2 (en) 2015-02-24 2022-03-15 10X Genomics, Inc. Methods and compositions for targeted nucleic acid sequence coverage
US10774370B2 (en) 2015-12-04 2020-09-15 10X Genomics, Inc. Methods and compositions for nucleic acid analysis
US11473125B2 (en) 2015-12-04 2022-10-18 10X Genomics, Inc. Methods and compositions for nucleic acid analysis
US11873528B2 (en) 2015-12-04 2024-01-16 10X Genomics, Inc. Methods and compositions for nucleic acid analysis
US11624085B2 (en) 2015-12-04 2023-04-11 10X Genomics, Inc. Methods and compositions for nucleic acid analysis
US11084036B2 (en) 2016-05-13 2021-08-10 10X Genomics, Inc. Microfluidic systems and methods of use
CN109790535A (en) * 2016-07-29 2019-05-21 新英格兰生物实验室公司 Prevent the method and composition of concatemerization during template switch
US11795492B2 (en) 2016-09-15 2023-10-24 ArcherDX, LLC. Methods of nucleic acid sample preparation
US11390905B2 (en) 2016-09-15 2022-07-19 Archerdx, Llc Methods of nucleic acid sample preparation for analysis of DNA
US10815525B2 (en) 2016-12-22 2020-10-27 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10011872B1 (en) 2016-12-22 2018-07-03 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10550429B2 (en) 2016-12-22 2020-02-04 10X Genomics, Inc. Methods and systems for processing polynucleotides
US11180805B2 (en) 2016-12-22 2021-11-23 10X Genomics, Inc Methods and systems for processing polynucleotides
US10480029B2 (en) 2016-12-22 2019-11-19 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10323278B2 (en) 2016-12-22 2019-06-18 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10793905B2 (en) 2016-12-22 2020-10-06 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10858702B2 (en) 2016-12-22 2020-12-08 10X Genomics, Inc. Methods and systems for processing polynucleotides
US11193122B2 (en) 2017-01-30 2021-12-07 10X Genomics, Inc. Methods and systems for droplet-based single cell barcoding
US10428326B2 (en) 2017-01-30 2019-10-01 10X Genomics, Inc. Methods and systems for droplet-based single cell barcoding
US11773389B2 (en) 2017-05-26 2023-10-03 10X Genomics, Inc. Single cell analysis of transposase accessible chromatin
US10844372B2 (en) 2017-05-26 2020-11-24 10X Genomics, Inc. Single cell analysis of transposase accessible chromatin
US10927370B2 (en) 2017-05-26 2021-02-23 10X Genomics, Inc. Single cell analysis of transposase accessible chromatin
US10400235B2 (en) 2017-05-26 2019-09-03 10X Genomics, Inc. Single cell analysis of transposase accessible chromatin
US11155810B2 (en) 2017-05-26 2021-10-26 10X Genomics, Inc. Single cell analysis of transposase accessible chromatin
US11198866B2 (en) 2017-05-26 2021-12-14 10X Genomics, Inc. Single cell analysis of transposase accessible chromatin
US11241688B2 (en) 2017-06-05 2022-02-08 10X Genomics, Inc. Gaskets for the distribution of pressures in a microfluidic system
US10876147B2 (en) 2017-11-15 2020-12-29 10X Genomics, Inc. Functionalized gel beads
US10745742B2 (en) 2017-11-15 2020-08-18 10X Genomics, Inc. Functionalized gel beads
US11884962B2 (en) 2017-11-15 2024-01-30 10X Genomics, Inc. Functionalized gel beads
US10829815B2 (en) 2017-11-17 2020-11-10 10X Genomics, Inc. Methods and systems for associating physical and genetic properties of biological particles
US11155881B2 (en) 2018-04-06 2021-10-26 10X Genomics, Inc. Systems and methods for quality control in single cell processing

Also Published As

Publication number Publication date
WO2000070095A3 (en) 2001-08-02

Similar Documents

Publication Publication Date Title
WO2000070095A2 (en) Homogeneous isothermal amplification and detection of nucleic acids using a template switch oligonucleotide
EP0904412B1 (en) Method for polynucleotide amplification
US6013439A (en) Detection of differences in nucleic acids
US6087133A (en) Isothermal strand displacement nucleic acid amplification
US6280952B1 (en) Two-step hybridization and capture of a polynucleotide
EP0876510A1 (en) Homogeneous amplification and detection of nucleic acids
EP1147230B1 (en) Method for controlling the extension of an oligonucleotide
US7105318B2 (en) Specific and sensitive nucleic acid detection method
JPH11510369A (en) Detection of nucleic acids by forming template-dependent products
WO1995016055A1 (en) Solution phase nucleic acid sandwich assays having reduced background noise
EP1210457B1 (en) Detection of differences in nucleic acids by inhibition of spontaneous dna branch migration
US20020182615A1 (en) All in one nucleic acid amplification assay
AU724804B2 (en) Assay involving looped nucleic acid
WO2000043543A1 (en) Detection of differences between polynucleotides
JP4104657B2 (en) Positive control for polynucleotide amplification
US20020031776A1 (en) Enzymatic labeling and detection of DNA hybridization probes
WO2000043546A9 (en) Detection of drug resistant organisms
WO2000046232A9 (en) Primer extension methods for production of high specific activity nucleic acid probes

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): CA JP

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
AK Designated states

Kind code of ref document: A3

Designated state(s): CA JP

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE