EP1583840A2 - Transcription dependante de la cible - Google Patents

Transcription dependante de la cible

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Publication number
EP1583840A2
EP1583840A2 EP03814301A EP03814301A EP1583840A2 EP 1583840 A2 EP1583840 A2 EP 1583840A2 EP 03814301 A EP03814301 A EP 03814301A EP 03814301 A EP03814301 A EP 03814301A EP 1583840 A2 EP1583840 A2 EP 1583840A2
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EP
European Patent Office
Prior art keywords
target
sequence
nucleic acid
complementary
probe
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP03814301A
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German (de)
English (en)
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EP1583840A4 (fr
Inventor
designation of the inventor has not yet been filed The
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
JENDRISAK Jerome J
Meis Judy E
Vaidyanathan Ramesh
Epicentre Technologies Corp
Original Assignee
JENDRISAK Jerome J
Meis Judy E
Vaidyanathan Ramesh
Epicentre Technologies Corp
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Application filed by JENDRISAK Jerome J, Meis Judy E, Vaidyanathan Ramesh, Epicentre Technologies Corp filed Critical JENDRISAK Jerome J
Publication of EP1583840A2 publication Critical patent/EP1583840A2/fr
Publication of EP1583840A4 publication Critical patent/EP1583840A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6862—Ligase chain reaction [LCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6865—Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/682—Signal amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to novel methods, compositions and kits for amplifying, detecting and quantifying one or multiple target nucleic acid sequences in a sample, including target sequences that differ by as little as one nucleotide.
  • the invention has broad applicability for research, environmental and genetic screening, and diagnostic applications, such as for detecting and quantifying sequences that indicate the presence of a pathogen, the presence of a gene or an allele, or the presence of a single nucleotide polymorphism (SNP) or other type of gene mutation or variant.
  • SNP single nucleotide polymorphism
  • the invention also related to novel methods, compositions and kits for detecting and quantifying abroad range of non-nucleic acid analytes by detecting a target sequence that is joined to an analyte-binding substance.
  • the promoter region contains a sequence of bases that signals RNA polymerase to associate with the DNA, and to initiate the transcription of mRNA using one ofthe DNA strands as a template to make a corresponding complementary strand of RNA.
  • RNA polymerases from different species typically recognize promoter regions comprised of different sequences.
  • the promoter driving transcription ofthe gene or DNA sequence must be a cognate promoter for the RNA polymerase, meaning that it is recognized by the RNA polymerase.
  • the presence of a nucleic acid sequence can indicate, for example, the presence of a pathogen, or the presence of particular genes or mutations in particular genes that correlate with or that are indicative ofthe presence or status of a disease state, such as, but not limited to, a cancer.
  • Still other methods use in vitro transcription as part of a process for amplifying and detecting one or more target nucleic acid sequences in order to detect the presence of a pathogen, such as a viral or microbial pathogen, that is a causative agent for a disease or to detect a gene sequence that is related to a disease or the status of a disease for medical purposes.
  • a pathogen such as a viral or microbial pathogen
  • Examples of methods that use in vitro transcription for this purpose include U.S. Patent Nos.
  • Still other methods detect sequences or mutations using methods that involve ligation of adjacently hybridizing oligonucleotide probes or ligation of non-adjacently hybridizing probes following a process such as primer extension.
  • Ligation detection methods include those disclosed in European Patent Application Publication Nos. 0246864 A2 and 0246864 Bl of Carr; U.S. Patent Nos. 4,883,750; 5,242,794; 5,521,065; 5,962,223; and 6,054,266 of Whiteley, N.M. et al.; U.S. Patent Nos. 4,988,617 of Landegren and Hood; U.S. Patent No. 5,871,921 of Landegren and Kwiatkowski; U.S.
  • Patent No. 5,866,337 of Schon European Patent Application Publication Nos. 0320308 A2 and 0320308 Bl ofBackman and Wang; PCT Publication No. WO 89/09835 of Orgel and Watt and European Patent Publication No. 0336731 Bl of Bruce Wallace; U.S. Patent No. 5,686,272 of Marshall et al.; U.S. Patent No. 5,869,252 of Bouma et al.; U.S. Patent Nos. 5,494,810; 5,830,711; 6,054,564; 6,027,889; 6,268,148; and 6,312,892 of Barany et al.; U.S. Patent Nos. 5,912,148 and 6,130,073 of F.
  • U.S. Patent No. 6,153,384 of Lynch et al. discloses an assay to identify ligase activity modulators by ligation of a labeled nucleic acid to an immobilized capture nucleic acid in the presence of a potential ligase activity modulator and U.S. Patent No. 5,976,806 of Mahajan et al. discloses a quantitative and functional DNA ligase assay that uses a linearized plasmid containing a reporter gene, wherein ligase activity is followed by the extent of coupled transcription-translation ofthe reporter gene.
  • U.S. Patent No. 5,807,674 of Sanjay Tyagi discloses detection of RNA target sequences by ligation ofthe RNA binary probes, wherein a substrate for Q-beta replicase is generated.
  • Japanese Patent Nos. JP4304900 and JP4262799 of Aono Toshiya et al. disclose detection of a target sequence by ligation of a linear single-stranded probe having target- complementary 3'- and 5'-end sequences which are adjacent when the linear probe is annealed to a target sequence in the sample, followed by either rolling circle replication or in vitro transcription ofthe circular single-stranded template.
  • the inventors disclose that in vitro transcription is performed by first annealing to the circular single-stranded template a complementary nucleotide primer having an anti-promoter sequence in order to form a double- stranded promoter, and then transcribing the circular single-stranded template having the annealed anti-promoter primer with an RNA polymerase that has helicase-like activity, such as T7, T3 or SP6 RNA polymerase.
  • RNA polymerase protopromoters in circular probe so that tandem-sequence single-stranded protopromoter- containing DNA products resulting from rolling circle replication can be transcribed by a cognate T7-type RNA polymerase following conversion of said DNA products to a form containing double-stranded promoters.
  • Rolling circle transcription of these circular ssDNAs occurs in the absence of primers, in the absence of a canonical promoter sequence, and in the absence of any duplex DNA structure, and results in synthesis of linear multimeric complementary copies ofthe circle sequence up to thousands of nucleotides in length. Transcription ofthe linear precursor ofthe circular ssDNA template yielded only a small amount of RNA transcript product that was shorter than the template. ⁇
  • nucleic acid amplification methods have been described in the art, there is a continuing need for methods and assays for detecting nucleic acids that are specific and accurate, yet are easier and faster than current methods.
  • the present invention provides novel assays, methods, compositions and kits that are simple in format and very rapid to perform, but that can be used to detect and quantify any of a broad range of analytes with a high degree of specificity and sensitivity, including both nucleic acid analytes and non-nucleic acid analytes.
  • the invention provides assays, methods and kits that can detect and distinguish between target sequences, including sequences that differ even by only a single nucleotide, such as for analysis of single nucleotide polymorphisms.
  • One embodiment ofthe invention is a method for detecting a target nucleic acid sequence, the method comprising: (a) providing one or more target probes comprising linear single-stranded DNA, the target probes comprising at least two target-complementary sequences that are not joined to each other, wherein the 5 '-end of a first target-complementary sequence is complementary to the 5 '-end ofthe target nucleic acid sequence, and wherein the 3 '-end of a second target-complementary sequence is complementary to the 3 '-end ofthe target nucleic acid sequence, and wherein the 3'-end ofthe first target-complementary sequence is joined to the 5'- end of a sense promoter sequence for an RNA polymerase; (b) contacting the target probes with the target nucleic acid sequence and incubating under hybridization conditions, wherein the target-complementary sequences anneal adjacently on the target nucleic acid sequence to form a complex; (c) contacting the complex with a ligas
  • the ligase has little or no activity in ligating blunt ends and is substantially more active in ligating ends that are adjacent when annealed to two contiguous regions of a target sequence compared to ends that are not annealed to the target sequence.
  • One suitable ligase that can be used is Ampligase® Thermostable DNA Ligase (EPICENTRE Technologies, Madison, WI).
  • the RNA polymerase comprises T7 RNAP, T3 RNAP, SP6 RNAP or another T7-like RNA polymerase or a mutant form of one of these T7-like RNA polymerases.
  • the anti-sense promoter oligo is attached to a solid support.
  • the anti-sense promoter oligo comprises a moiety, such as, but not limited to a biotin moiety that permits binding ofthe anti-sense promoter oligo to a solid support after annealing to the ligation product to obtain a transcription substrate.
  • the target probes comprise monopartite target probes comprising a promoter target probe and a signal target probe and or optionally, one or more simple target probes. In other embodiments, a bipartite target probe and, optionally, one or more simple target probes is used.
  • the target sequence comprises a target nucleic acid in a sample
  • the target sequence comprises a target sequence tag that is joined to an analyte-binding substance that binds an analyte in the sample.
  • the transcription substrate that is transcribed remains catenated to a target nucleic acid.
  • the target sequence is preferably less than about 150 to about 200 nucleotides from the 3'-end ofthe target nucleic acid or target sequence tag.
  • one or more additional steps is used in order to release the catenated circular ligation product from the target sequence prior to transcription, as described elsewhere herein.
  • Another embodiment ofthe invention comprises a method for obtaining transcription products comprising multiple copies of a target nucleic acid sequence in a sample, said method comprising: (a) providing one or more target probes comprising linear single- stranded DNA, the target probes having at least two different target-complementary sequences that are not joined to each other, wherein the 5'-end of a first target-complementary sequence is complementary to the 5 '-end ofthe target nucleic acid sequence and the 3 '-end of a second target-complementary sequence is complementary to the 3'-end ofthe target nucleic acid sequence, and wherein the target probe that comprises a target-complementary sequence that is complementary to the 5'-end ofthe target nucleic acid sequence also comprises a sense promoter sequence that is joined to the 3'-end ofthe target-complementary sequence of said target probe, and wherein any additional target probes, if provided, comprise simple target probes having target-complementary sequences that anneal to the target nucleic acid sequence
  • the ligase has little or no activity in ligating blunt ends and is substantially more active in ligating ends that are adjacent when annealed to two contiguous regions of a target sequence compared to ends that are not annealed to the target sequence.
  • One suitable ligase that can be used is Ampligase® Thermostable DNA Ligase (EPICENTRE Technologies, Madison, WI).
  • Some preferred RNA polymerases are T7 RNAP, T3 RNAP, SP6 RNAP or another T7-like RNA polymerase or a mutant form of one of these T7-like RNA polymerases.
  • the anti-sense promoter oligo is attached to a solid support.
  • the anti-sense promoter oligo comprises a moiety, such as, but not limited to a biotin moiety that permits binding ofthe anti-sense promoter oligo to a solid support after annealing to the ligation product to obtain a transcription substrate.
  • the target probes comprise monopartite target probes comprising a promoter target probe and a signal target probe and/or optionally, one or more simple target probes. In other embodiments, a bipartite target probe and, optionally, one or more simple target probes is used.
  • the target sequence comprises a target nucleic acid in a sample
  • the target sequence comprises a target sequence tag that is joined to an analyte-binding substance that binds an analyte in the sample.
  • the transcription substrate that is transcribed remains catenated to a target nucleic acid.
  • the target sequence is preferably less than about 150 to about 200 nucleotides from the 3 '-end ofthe target nucleic acid or target sequence tag.
  • one or more additional steps is used in order to release the catenated circular ligation product from the target sequence prior to transcription, as described elsewhere herein.
  • Another embodiment ofthe present invention comprises a method for obtaining a transcription product complementary to a target nucleic acid sequence (target sequence or target), said method comprising: (a) providing a target sequence amplification probe (TSA probe), wherein said TS probe comprises a linear single-stranded DNA comprising two end portions that are not joined, which end portions are connected by an intervening sequence, wherein the 5 '-end target-complementary sequence is complementary to the 5 '-end ofthe target sequence, and wherein the 3 '-end target-complementary sequence is complementary to the 3'- end ofthe target sequence, and wherein joining ofthe ends of said TSA probe forms a TSA circle; (b) contacting the TSA probe to the target sequence and incubating under hybridization conditions, wherein the target-complementary sequences anneal adjacently on the target sequence; (c) contacting said TSA probe annealed to said target sequence with a ligase under ligation conditions so as to obtain a TSA circle; (d)
  • the target probes comprise a bipartite target probe and optionally, one or more simple target probes. In other embodiments, the target probes comprise monopartite target probes comprising a promoter target probe and a signal target probe and/or one or more simple target probes.
  • the ligase has little or no activity in ligating blunt ends and is substantially more active in ligating ends that are adjacent when annealed to two contiguous regions of a target sequence compared to ends that are not annealed to the target sequence.
  • One suitable ligase that can be used is Ampligase® Thermostable DNA Ligase (EPICENTRE Technologies, Madison, WI).
  • a preferred strand-displacing DNA polymerase that can be used is IsoThermTM DNA Polymerase (EPICENTRE Technologies, Madison, WI).
  • RNA polymerases are T7 RNAP, T3 RNAP, SP6 RNAP or another T7-like RNA polymerase or a mutant form of one of these T7- like RNA polymerases.
  • AmpliScribe T7-FlashTM Transcription Kit is used for in vitro transcription ofthe transcription substrate (EPICENTRE Technologies, Madison, WI).
  • the anti-sense promoter oligo is attached to a solid support.
  • the anti-sense promoter oligo comprises a moiety, such as, but not limited to a biotin moiety that permits binding ofthe anti-sense promoter oligo to a solid support after annealing to the ligation product to obtain a transcription substrate.
  • the target sequence comprises a target nucleic acid in a sample, whereas in other embodiments the target sequence comprises a target sequence tag that is joined to an analyte- binding substance that binds an analyte in the sample.
  • the TSA circle or circular transcription substrate, respectively remains catenated to a target nucleic acid.
  • the target sequence is preferably less than about 150 to about 200 nucleotides from the 3'-end ofthe target nucleic acid or target sequence tag. In still other embodiments of methods in which the target sequence is greater than about 150 to about 200 nucleotides from the 3 '-end ofthe target nucleic acid or target sequence tag, then one or more additional steps is used in order to release the catenated TSA circles from the target sequence prior to rolling circle replication, as described elsewhere herein.
  • one or more additional steps is used in order to release the catenated circular ssDNA ligation products that result from ligation of bipartite target probes that are annealed to target sequences in the rolling circle replication product more than about 150 nucleotides to about 200 nucleotides from the 3 '-end of to the rolling circle replication product.
  • Yet another embodiment is a method for detecting a target sequence, said method comprising: (a) providing a first bipartite target probe comprising linear single-stranded DNA having two target-complementary sequences that are not joined to each other and that are contiguous when annealed to the target sequence, wherein the 5'-end of he first target- complementary sequence is complementary to the 5 '-end ofthe target nucleic acid sequence, and wherein the 3 '-end of a second target-complementary sequence is complementary to the 3'- end ofthe target nucleic acid sequence, and wherein the 3'-end ofthe first target-complementary sequence is joined to the 5 '-end of a sense promoter sequence for an RNA polymerase; (b) providing a second bipartite target probe comprising linear single-stranded DNA having two end sequences that are not joined to each other and that, when joined, are identical to the target sequence, wherein the 5 '-end ofthe first end sequence is complementary to the target- complementary
  • the target sequence is less than about 150 to about 200 nucleotides from the 3 '-end ofthe target nucleic acid or target sequence tag.
  • the target sequence is greater than about 150 to about 200 nucleotides from the 3'-end ofthe target nucleic acid or target sequence tag, one or more additional steps is used in order to release the catenated circular ligation products from the target sequence when the target probe anneals to a sequence in a linear DNA molecule that is greater than about 150 to about 200 nucleotides from the 3 '-end of the linear DNA molecule.
  • circular transcription substrates that are transcribed remain catenated to a target nucleic acid.
  • the ligase has little or no activity in ligating blunt ends and is substantially more active in ligating ends that are adjacent when annealed to a contiguous complementary sequence compared to ends that are not adjacently annealed to acomplementary sequence.
  • One suitable ligase that can be used is Ampligase® Thermostable DNA Ligase (EPICENTRE Technologies, Madison, WI).
  • Suitable reverse transcriptases that can be used are MMLV Reverse Transcriptase or IsoThermTM DNA Polymerase (EPICENTRE Technologies, Madison, WI).
  • RNA polymerases are T7 RNAP, T3 RNAP, SP6 RNAP or another T7-like RNA polymerase or a mutant form of one of these T7-like RNA polymerases.
  • AmpliScribeTM T7 -FlashTM Transcription Kit is used for in vitro transcription ofthe transcription substrate (EPICENTRE Technologies, Madison, WI).
  • the anti-sense promoter oligo is attached to a solid support.
  • the anti-sense promoter oligo comprises a moiety, such as, but not limited to a biotin moiety that permits binding ofthe anti-sense promoter oligo to a solid support after annealing to the ligation product to obtain a transcription substrate.
  • the target sequence comprises a target nucleic acid in a sample, whereas in other embodiments the target sequence comprises a target sequence tag that is joined to an analyte- binding substance that binds an analyte in the sample.
  • the present invention also comprises additional embodiments, described below, that use an RNA polymerase that recognizes a cognate single-stranded transcription promoter or a single-stranded pseudopromoter.
  • the ability to use a single-sfranded promoter or pseudopromoter simplifies an assay or method ofthe invention since a transcription substrate can be obtained without the need to complex an anti- sense promoter oligo with a sense promoter sequence in a product of ligation of one or more target probes annealed to a target sequence.
  • these embodiments obviously cannot use methods that comprise annealing to an anti-sense promoter oligo that is attached to a solid support.
  • one embodiment comprises a method to detect a target nucleic acid sequence, the method comprising a DNA ligation operation and a transcription operation, wherein the DNA ligation operation comprises ligation of one or more target probes comprising a promoter that that binds an RNA polymerase that can bind a single-stranded promoter and initiate transcription therefrom, wherein the ligation is dependent on hybridization ofthe target probes to the target nucleic acid sequence, and wherein the transcription operation comprises contacting the transcription substrate with an RNA polymerase that binds the single-stranded promoter under transcription condition to obtain a transcription product.
  • the ligase has little or no activity in ligating blunt ends and is substantially more active in ligating ends that are adjacent when annealed to two contiguous regions of a target sequence compared to ends that are not annealed to the target sequence.
  • One suitable ligase that can be used is Ampligase® Thermostable DNA Ligase (EPICENTRE Technologies, Madison, WI).
  • the single-stranded promoter comprises an N4 RNAP promoter and the RNA polymerase comprises an N4 mini-vRNAP enzyme, which comprises a transcriptionally active 1,106-amino acid domain corresponding to amino acids 998-2103 of N4 vRNAP, or a mutant form of N4 mini-vRNAP that comprises a mutation at position number Y678.
  • the single-stranded promoter comprises a pseudopromoter for a T7-type RNAP (e.g., T7 RNAP, T3 RNAP or SP6 RNAP), E. coli RNAP or a Thermus RNAP, and the cognate RNA polymerase for the promoter is used.
  • a bipartite target probe and, optionally, one or more simple target probes is used.
  • the target probes comprise monopartite target probes comprising a promoter target probe and a signal target probe and/or optionally, one or more simple target probes.
  • the target sequence comprises a target nucleic acid in a sample, whereas in other embodiments the target sequence comprises a target sequence tag that is joined to an analyte-binding substance that binds an analyte in the sample.
  • the transcription substrate that is transcribed remains catenated to a target nucleic acid.
  • the target sequence is preferably less than about 150 to about 200 nucleotides from the 3 '-end ofthe target nucleic acid or target sequence tag.
  • the target sequence is greater than about 150 to about 200 nucleotides from the 3'-end ofthe target nucleic acid or target sequence tag comprising the target sequence, one or more additional steps is used in order to release the catenated circular ligation product from the target sequence prior to transcription, as described elsewhere herein.
  • One aspect of this embodiment ofthe invention comprises a method for detecting a target nucleic acid sequence, the method comprising: (a) providing one or more target probes comprising linear single-stranded DNA, the target probes comprising at least two target- complementary sequences that are not joined to each other, wherein the 5 '-end of a first target- complementary sequence is complementary to the 5 '-end ofthe target nucleic acid sequence, and wherein the 3 '-end of a second target-complementary sequence is complementary to the 3'- end ofthe target nucleic acid sequence, and wherein the target probe that comprises the first target-complementary sequence also comprises a promoter that is joined to the 3'-end ofthe first target complementary sequence, which promoter can bind a single-stranded promoter and initiate transcription therefrom; (b) contacting the target probes with the target nucleic acid sequence and incubating under hybridization conditions, wherein the target-complementary sequences anneal adjacently to the target nucleic acid sequence to form
  • the ligase has little or no activity in ligating blunt ends and is substantially more active in ligating ends that are adjacent when annealed to two contiguous regions of a target sequence compared to ends that are not annealed to the target sequence.
  • One suitable ligase that can be used is Ampligase® Thermostable DNA Ligase (EPICENTRE Technologies, Madison, WI).
  • the single-stranded promoter comprises an N4 RNAP promoter and the RNA polymerase comprises an N4 mini-vRNAP enzyme, which comprises a transcriptionally active 1,106-amino acid domain corresponding to amino acids 998-2103 of N4 vRNAP, or a mutant form of N4 mini-vRNAP that comprises a mutation at position number Y678.
  • the single-stranded promoter comprises a pseudopromoter for a T7-type RNAP (e.g., T7 RNAP, T3 RNAP or SP6 RNAP), E. coli RNAP or a Thermus RNAP, and the cognate RNA polymerase for the promoter is used.
  • a bipartite target probe and, optionally, one or more simple target probes is used.
  • the target probes comprise monopartite target probes comprising a promoter target probe and a signal target probe and/or optionally, one or more simple target probes.
  • the target sequence comprises a target nucleic acid in a sample, whereas in other embodiments the target sequence comprises a target sequence tag that is joined to an analyte-binding substance that binds an analyte in the sample.
  • the transcription substrate that is transcribed remains catenated to a target nucleic acid.
  • the target sequence is preferably less than about 150 to about 200 nucleotides from the 3'-end ofthe target nucleic acid or target sequence tag.
  • the target sequence is greater than about 150 to about 200 nucleotides from the 3'-end ofthe target nucleic acid or target sequence tag comprising the target sequence, one or more additional steps is used in order to release the catenated circular ligation product from the target sequence prior to transcription, as described elsewhere herein.
  • Another aspect of this embodiment of he invention comprises a method for detecting a target nucleic acid sequence, the method comprising: (a) providing one or more target probes comprising linear single-stranded DNA, the target probes comprising at least two target-complementary sequences that are not joined to each other, wherein the 5 '-end of a first target-complementary sequence is complementary to the 5 '-end ofthe target nucleic acid sequence, and wherein the 3 '-end of a second target-complementary sequence is complementary to the 3 '-end ofthe target nucleic acid sequence, and wherein the target probe that comprises the first target- complementary sequence also comprises a promoter that is joined to the 3 '-end of the first target-complementary sequence, which promoter binds an RNA polymerase that can bind a single-stranded promoter and initiate transcription therefrom; (b) contacting the target probes with the target nucleic acid sequence and incubating under hybridization conditions whereby the target probes anne
  • the ligase has little or no activity in ligating blunt ends and is substantially more active in ligating ends that are adjacent when annealed to two contiguous regions of a target sequence compared to ends that are not annealed to the target sequence.
  • One suitable ligase that can be used is Ampligase® Thermostable DNA Ligase (EPICENTRE Technologies, Madison, WI).
  • the single-stranded promoter comprises an N4 RNAP promoter and the RNA polymerase comprises an N4 mini-vRNAP enzyme, which comprises a transcriptionally active 1,106-amino acid domain corresponding to amino acids 998-2103 of N4 vRNAP, or a mutant form of N4 ini- vRNAP that comprises a mutation at position number Y678.
  • the single- stranded promoter comprises a pseudopromoter for a T7-type RNAP (e.g., T7 RNAP, T3 RNAP or SP6 RNAP), E.
  • the target sequence comprises a target nucleic acid in a sample, whereas in other embodiments the target sequence comprises a target sequence tag that is joined to an analyte-binding substance that binds an analyte in the sample.
  • the transcription substrate that is transcribed remains catenated to a target nucleic acid.
  • the target sequence is preferably less than about 150 to about 200 nucleotides from the 3 '-end ofthe target nucleic acid or target sequence tag.
  • one or more additional steps is used in order to release the catenated circular ligation product from the target sequence prior to transcription, as described elsewhere herein.
  • Another embodiment ofthe invention comprises a method for obtaining transcription products comprising multiple copies of a target nucleic acid sequence (target sequence) in a sample, said method comprising: (a) providing one or more target probes comprising linear single-stranded DNA, said one or more target probes having at least two different target-complementary sequences that are not joined to each other, wherein the 5'-end of a first target-complementary sequence is complementary to the 5'-end ofthe target sequenbe and the 3'-end of a second target-complementary sequence is complementary to the 3'-end ofthe target sequence, and wherein the target probe that comprises a target-complementary sequence that is complementary to the 5'-end ofthe target sequence also comprises a promoter that is 3'-of the target-complementary sequence of said target probe, which promoter is for an RNA polymerase that lacks helicase-like activity and that can bind said single-stranded promoter and initiate transcription therefrom under transcription conditions, and wherein any additional target probe
  • the ligase has little or no activity in ligating blunt ends and is substantially more active in ligating ends that are adjacent when annealed to two contiguous regions of a target sequence compared to ends that are not annealed to the target sequence.
  • One suitable ligase that can be used is Ampligase® Thermostable DNA Ligase (EPICENTRE Technologies, Madison, WI).
  • the single-stranded promoter comprises an N4 RNAP promoter and the RNA polymerase comprises an N4 mini-vRNAP enzyme, which comprises a transcriptionally active 1,106-amino acid domain corresponding to amino acids 998-2103 of N4 vRNAP, or a mutant form of N4 mini-vRNAP that comprises a mutation at position number Y678.
  • the single-stranded promoter comprises a pseudopromoter for a T7-type RNAP (e.g., T7 RNAP, T3 RNAP or SP6 RNAP), E. coli RNAP or a Thermus RNAP, and the cognate RNA polymerase for the promoter is used.
  • a bipartite target probe and, optionally, one or more simple target probes is used.
  • the target probes comprise monopartite target probes comprising a promoter target probe and a signal target probe and/or optionally, one or more simple target probes.
  • the target sequence comprises a target nucleic acid in a sample, whereas in other embodiments the target sequence comprises a target sequence tag that is joined to an analyte-binding substance that binds an analyte in the sample.
  • the transcription substrate that is transcribed remains catenated to a target nucleic acid.
  • the target sequence is preferably less than about 150 to about 200 nucleotides from the 3 '-end ofthe target nucleic acid or target sequence tag.
  • the target sequence is greater than about 150 to about 200 nucleotides from the 3'-end ofthe target nucleic acid or target sequence tag comprising the target sequence, one or more additional steps is used in order to release the catenated circular ligation product from the target sequence prior to transcription, as described elsewhere herein.
  • Another embodiment ofthe present invention comprises a method for obtaining a transcription product complementary to a target nucleic acid sequence (target sequence or target), said method comprising: (a) providing a target sequence amplification probe (TS probe), wherein said TSA probe comprises a linear single-stranded DNA comprising two end portions that are not joined, which end portions are connected by an intervening sequence, wherein the 5 '-end target-complementary sequence is complementary to the 5 '-end ofthe target sequence, and wherein the 3 '-end target-complementary sequence is complementary to the 3'- end ofthe target sequence, and wherein joining ofthe ends of said TSA probe forms a TSA circle; (b) contacting the TSA probe to the target sequence and incubating under hybridization conditions, wherein the target-complementary sequences anneal adjacently on the target sequence; (c) contacting said TSA probe annealed to said target sequence with a ligase under ligation conditions so as to obtain a TSA circle; (d) providing a target
  • the target probes comprise a bipartite target probe and optionally, one or more simple target probes. In other embodiments, the target probes comprise monopartite target probes comprising a promoter target probe and a signal target probe and/or one or more simple target probes.
  • only one ligase is used for ligating both the TSA probe and the target probes.
  • the ligase has little or no activity in ligating blunt ends and is substantially more active in ligating ends that are adjacent when annealed to two contiguous regions of a target sequence compared to ends that are not annealed to the target sequence.
  • One suitable ligase that can be used is Ampligase® Thermostable DNA Ligase (EPICENTRE Technologies, Madison, WI).
  • a preferred strand-displacing DNA polymerase that can be used is IsoThermTM DNA Polymerase (EPICENTRE Technologies, Madison, WI).
  • the single-stranded promoter comprises an N4 RNAP promoter and the RNA polymerase comprises an N4 mini-vRNAP enzyme, which comprises a transcriptionally active 1,106-amino acid domain corresponding to amino acids 998-2103 of N4 vRNAP, or a mutant form of N4 mini- vRNAP that comprises a mutation at position number Y678.
  • the single- stranded promoter comprises a pseudopromoter for a T7-type RNAP (e.g., T7 RNAP, T3 RNAP or SP6 RNAP), E. coli RNAP or a Thermus RNAP, and the cognate RNA polymerase for the promoter is used.
  • a bipartite target probe and, optionally, one or more simple target probes is used.
  • the target probes comprise monopartite target probes comprising a promoter target probe and a signal target probe and/or optionally, one or more simple target probes.
  • the target sequence comprises a target nucleic acid in a sample
  • the target sequence comprises a target sequence tag that is joined to an analyte-binding substance that binds an analyte in the sample.
  • the TSA circle or circular transcription substrate, respectively remains catenated to a target nucleic acid.
  • the target sequence is preferably less than about 150 to about 200 nucleotides from the 3'-end ofthe target nucleic acid or target sequence tag.
  • one or more additional steps is used in order to release the catenated TSA circles from the target sequence prior to rolling circle replication, as described elsewhere herein.
  • one or more additional steps can be used in order to release the catenated circular ssD ligation products that result from ligation of bipartite target probes that are annealed to target sequences in the rolling circle replication product more than about 150 nucleotides to about 200 nucleotides from the 3'-end of to the rolling circle replication product.
  • rolling circle replication is carried out using a ratio of dUTP to dTTP that results in incorporation of a dUMP residue about every 100-400 nucleotides and a composition comprising uracil-N-glycosylase and endonuclease IV is used to release catenated DNA molecules that are ligated on the linear rolling circle replication product following annealing of bipartite target probes to the replicated target sequences.
  • Yet another embodiment is a method for detecting a target sequence, said method comprising: (a) providing a first bipartite target probe, wherein said first bipartite target probe comprises a 5 '-portion and a 3 '-portion, wherein said 5 '-portion comprises: (i) a 5 '-end portion that comprises a sequence that is complementary to a target sequence, and (ii) a promoter sequence, wherein said promoter sequence is covalently attached to and 3 '-of said target- complementary sequence in said 5 '-portion; and wherein said 3 '-portion comprises: (i) a 3 '-end portion that comprises a sequence that is complementary to a target sequence, wherein said target-complementary sequence of said 3 '-end portion, when annealed to said target sequence, is adjacent to said target-complementary sequence of said 5 '-end portion of said first bipartite target probe, and (ii) optionally, a signal sequence, wherein said signal sequence is 5 '-end
  • one or more additional steps is used in order to release the catenated circular ligation products from the target sequence when the target probe anneals to a sequence in a linear DNA molecule that is greater than about 150 to about 200 nucleotides from the 3 '-end ofthe linear DNA molecule.
  • circular transcription substrates that are transcribed remain catenated to a target nucleic acid.
  • only one ligase is used for all ligation reactions.
  • the ligase has little or no activity in ligating blunt ends and is substantially more active in ligating ends that are adjacent when annealed to a contiguous complementary sequence compared to ends that are not adjacently annealed to acomplementary sequence.
  • One suitable ligase that can be used is Ampligase® Thermostable DNA Ligase (EPICENTRE Technologies, Madison, WI).
  • Suitable reverse transcriptases that can be used are MMLV Reverse Transcriptase or IsoThermTM DNA Polymerase (EPICENTRE Technologies, Madison, WI).
  • the single-stranded promoter comprises an N4 RNAP promoter and the RNA polymerase comprises an N4 mini-vRNAP enzyme, which comprises a transcriptionally active 1,106-amino acid domain corresponding to amino acids 998-2103 of N4 vRNAP, or a mutant form of N4 mini- vRNAP that comprises a mutation at position number Y678.
  • the single- stranded promoter comprises a pseudopromoter for a T7-type RNAP (e.g., T7 RNAP, T3 RNAP or SP6 RNAP), E. coli RNAP or a Tliermus RNAP, and the cognate RNA polymerase for the promoter is used.
  • One embodiment that uses a double-stranded promoter and generates a circular transcription substrate comprises a method for detecting a target nucleic acid sequence, said method comprising: (a) providing at least two simple target probes comprising at least two target-complementary sequences, wherein the target probes comprise a 5 '-phosphate and are adjacent when annealed on the target sequence, and wherein a first simple target probe is complementary to the 5'-end ofthe target nucleic acid and a second simple target probe is complementary to the 3'-end ofthe target nucleic acid sequence; (b) annealing the target probes to the target nucleic acid sequence under hybridization conditions; (c) contacting the target probes annealed to the target nucleic acid sequence with a ligase under ligation conditions so as to obtain a linear ligation product; (d) denaturing the ligation product from the target nucleic acid sequence;
  • Another embodiment that uses a double-stranded promoter and generates a linear transcription substrate comprises a method for detecting a target nucleic acid sequence, said method comprising: (a) providing at least two simple target probes comprising at least two target-complementary sequences, wherein the target probes comprise a 5'-phosphate and are adjacent when annealed on the target sequence, and wherein a first simple target probe is complementary to the 5'-end ofthe target nucleic acid and a second simple target probe is complementary to the 3'-end ofthe target nucleic acid sequence; (b) annealing the target probes to the target nucleic acid sequence under hybridization conditions; (c) contacting the target probes annealed to the target nucleic acid sequence with a ligase under ligation conditions so as to obtain a first linear ligation product; (d) denaturing the ligation product from the target nucleic acid sequence;
  • a promoter oligo comprising an oligodeoxyribonucleotide having a 5'-phosphate group and a sense promoter sequence for a double-stranded transcription promoter that is recognized by a cognate RNA polymerase;
  • a signal oligo comprising an oligodeoxyribonucleotide comprising a signal sequence;
  • One embodiment that uses a single-stranded promoter and generates a circular transcription substrate comprises a method for detecting a target nucleic acid sequence, said method comprising: (a) providing at least two simple target probes comprising at least two target-complementary sequences, wherein the target probes comprise a 5'-phosphate and are adjacent when annealed on the target sequence, and wherein a first simple target probe is complementary to the 5'-end ofthe target nucleic acid and a second simple target probe is complementary to the 3'-end ofthe target nucleic acid sequence; (b).
  • Another embodiment that uses a single-stranded promoter and generates a linear transcription substrate comprises a method for detecting a target nucleic acid sequence, said method comprising: (a) providing at least two simple target probes comprising at least two target-complementary sequences, wherein the target probes comprise a 5'- ⁇ hosphate and are adjacent when annealed on the target sequence, and wherein a first simple target probe is complementary to the 5'-end ofthe target nucleic acid and a second simple target probe is complementary to the 3'-end ofthe target nucleic acid sequence; (b) annealing the target probes to the target nucleic acid sequence under hybridization conditions; (c) contacting the target probes annealed to the target nucleic acid sequence with a ligase under ligation conditions so as to obtain a first linear ligation product; (d) denaturing the ligation product from the target nucleic acid sequence; [0042] (e) providing a promoter oligo comprising at least two
  • Still another embodiment ofthe invention is a method for detecting a target nucleic acid sequence, the method comprising: (a) providing a simple bipartite target probe comprising linear single-stranded DNA (ssDNA) that lacks a sequence for a known promoter for an RNA polymerase, the simple bipartite target probe comprising two target-complementary sequences that are not joined to each other, wherein the 5 '-end of a first target-complementary sequence is complementary to the 5 '-end ofthe target nucleic acid sequence, and wherein the 3'- end of a second target-complementary sequence is complementary to the 3 '-end ofthe target nucleic acid sequence, wherein said simple bipartite target probe is transcribed little or not at all by an RNA polymerase under conditions in which a circular ssDNA obtained by intramolecular ligation ofthe simple bipartite target probe is transcribed efficiently by said RNA polymerase;
  • ssDNA linear single-stranded DNA
  • the ligase has little or no activity in ligating blunt ends and is substantially more active in ligating ends that are adjacent when annealed to two contiguous regions of a target sequence compared to ends that are not annealed to the target sequence.
  • One suitable ligase that can be used is Ampligase® Thermostable DNA Ligase (EPICENTRE Technologies, Madison, WI).
  • the RNA polymerase comprises an RNA polymerase chosen from among a T7 RNAP, a T3 RNAP, an SP6 RNAP or another T7-like RNA polymerase, including mutant forms thereof, or E. coli RNA polymerase or Thermus thermophilus RNA polymerase.
  • Another suitable RNA polymerase is an N4 mini-vRNAP.
  • the target sequence comprises a target nucleic acid in a sample, whereas in other embodiments the target sequence comprises a target sequence tag that is joined to an analyte-binding substance that binds an analyte in the sample.
  • the method is used to detect a single-nucleotide polymo ⁇ hism (SNP) or mutation, in which case the 5 '-nucleotide ofthe first target-complementary sequence or the 3'- end ofthe second target-complementary sequence of said simple bipartite target probe is complementary to the intended target nucleotide ofthe target sequence, and ligation only occurs when the ends of both target-complementary sequences are adjacently annealed on the target sequence, including the target nucleotide, under the stringent ligation conditions ofthe assay or method.
  • the target sequence is preferably less than about 150 to about 200 nucleotides from the 3 '-end ofthe target nucleic acid or target sequence tag.
  • the target sequence is greater than about 150 to about 200 nucleotides from the 3'-end ofthe target nucleic acid or target sequence tag comprising the target sequence
  • one or more additional steps is used in order to release the catenated circular ligation product from the target sequence prior to transcription, as described elsewhere herein.
  • the circular transcription substrate that is transcribed remains catenated to a target nucleic acid.
  • Another embodiment ofthe present invention is a method for detecting an analyte in a sample, wherein said analyte comprises a biomolecule that is not a nucleic acid, said method comprising: (a) providing an analyte-binding substance comprising a nucleic acid, wherein said nucleic acid binds with selectivity and high affinity to said analyte; (b) providing target probes comprising either (i) a promoter target probe and one or more additional target probes chosen from among a signal target probe and simple target probe; or (ii) a bipartite target probe and, if said target-complementary sequences of said bipartite target probe are not contiguous when annealed to said target sequence in said analyte-binding substance, optionally, one or more simple target probes; wherein said target probes of (i) or (ii) comprise sequences that are complementary to adjacent regions of a target sequence in said analyte-binding substance; (
  • the invention also comprises methods, compositions and kits for using ssDNA transcription substrates and RNA polymerases that can transcribe said ssDNA transcription substrates as a signaling system for an analyte of any type, including analytes such as, but not limited to, antigens, antibodies or other substances, in addition to an analyte that is a target nucleic acid.
  • analytes such as, but not limited to, antigens, antibodies or other substances, in addition to an analyte that is a target nucleic acid.
  • the invention comprises a method for detecting an analyte in or from a sample, said method comprising: 1. providing a transcription signaling system, said franscription signaling system comprising a ssDNA comprising: (a) a 5 '-portion comprising a sense promoter sequence for a double-stranded promoter for a cognate RNA polymerase; and (b) a signal sequence, wherein said signal sequence, when transcribed by said RNA polymerase, is detectable in some manner; 2.
  • RNA polymerase synthesizes RNA that is complementary to said signal sequence in said ssDNA transcription signaling system under said transcription conditions; 7. obtaining the RNA synthesis product that is complementary to said signal sequence in said ssDNA transcription signaling system; and 8. detecting said RNA synthesis product or a substance that results from said RNA synthesis product.
  • Another embodiment ofthe present invention comprises a method for amplifying a target nucleic acid by strand displacement reverse transcription of a linear single-stranded RNA (ssRNA) template, said method comprising: 1. providing a reaction mixture comprising: (a) a reverse transcriptase with strand-displacement activity; (b) optionally, a single-strand binding protein; and (c) multiple oligonucleotide primers, wherein at least the 3 '-portion of each said primer comprises a sequence that is complementary to a sequence in said ssRNA; 2.
  • ssRNA linear single-stranded RNA
  • reaction mixture containing said sample is maintained at a temperature wherein said reverse transcriptase, and optionally said single-strand binding protein, are optimally active in combination for strand-displacement reverse transcription and wherein said reverse transcription primers anneal to said target sequence, if present, with specificity, and wherein said temperature of said reaction mixture is maintained for a time sufficient to permit synthesis of first-strand cDNA reverse transcription products complementary to said target nucleic acid, if present in said sample; and 3. obtaining multiple copies of said first-strand cDNA that is complementary to said RNA target nucleic acid.
  • Still another embodiment of the present invention comprises a method for amplifying a target nucleic acid comprising a circular single-stranded RNA (ssRNA) by strand displacement reverse transcription, said method comprising: 1. providing a reaction mixture comprising: (a) a reverse transcriptase with strand-displacement activity; (b) optionally, a single- strand binding protein; and (c) at least one, and optionally multiple oligonucleotide primers, wherein at least the 3 '-portion of each said primer comprises a sequence that is complementary to a sequence in said ssRNA; 2.
  • ssRNA circular single-stranded RNA
  • the primers in the above methods for strand displacement reverse transcription comprise DNA oligonucleotides.
  • the primers comprise ribonucleotides, and in still other embodiments the primers comprise 2'-fluoro- containing modified oligoribonucleotides or DuraScriptTM RNA, for example made using the DuraScribeTM Transcription Kit (EPICENTRE Technologies, Madison, WI, USA).
  • a primer for strand-displacement reverse transcription can comprise a specific sequence that is complementary to only one RNA sequence, or alternatively, the multiple strand-displacement primers of a strand-displacement reverse franscription reaction ofthe present invention can also comprise random sequence primers, including but not limited to random hexamers, random octamers, random nonamers, random decamers, or random dodecamers.
  • the primers can also prime synthesis of second-strand cDNA using first-strand cDNA as a template, and subsequently, can prime the synthesis of third, fourth and other cDNA strands, thereby resulting in additional amplification.
  • the random sequence primers comprise alpha-thio internucleoside linkages, which are resistant to some exonucleases.
  • a biotin or other binding moiety is covalently attached to a nucleotide in the 5 '-portion of a reverse transcription primer used for strand- displacement reverse transcription. The biotin or other binding moiety enables capture of first- strand cDNA obtained by strand-displacement reverse transcription.
  • Strand-displacing reverse transcriptases that can be used include, but are not limited to RNaseH-Minus MMLV reverse transcriptase or IsoThermTM DNA Polymerase (EPICENTRE Technologies, Madison, WI).
  • One reverse transcription reaction condition that can increase displacement of first-strand cDNA is addition of a single-strand binding protein, such as, but not limited to EcoSSB Protein or an SSB Protein from a thermostable bacterium, such as Tth or Bst SSB Protein, to a reverse transcription reaction.
  • a single-strand binding protein such as, but not limited to EcoSSB Protein or an SSB Protein from a thermostable bacterium, such as Tth or Bst SSB Protein
  • Betaine can also be added to a reverse transcription reaction in order to increase strand displacement.
  • a reverse transcription reaction As disclosed in U.S. Patent Nos. 6,048,696 and 6,030,814, and in German Patent No. DE4411588C1, all of which are incorporated herein by reference and made part of the present invention, it is prefe ⁇ ed in many embodiments to use a final concentration of about 0.25 M, about 0.5 M, about 1.0 M, about 1.5 M, about 2.0 M, about 2.5 M or between about 0.25 M and 2.5 M betaine (trimethylglycine) in DNA polymerase or reverse transcriptase reactions in order to decrease DNA polymerase stops and increase the specificity of reactions that use a DNA polymerase.
  • betaine trimethylglycine
  • FIG. 1 illustrates an example of different monopartite target probes and a related composition - an anti-sense promoter oligo - for embodiments ofthe invention that use a promoter target probe that comprises a sense promoter sequence for an RNA polymerase that uses a double-stranded promoter.
  • FIG. 2 shows an example of a bipartite target probe and a related composition - an anti-sense promoter oligo - for embodiments ofthe invention that use a bipartite target probe that comprises a sense promoter sequence for an RNA polymerase that uses a double-sfranded promoter.
  • FIG. 3 shows a basic embodiment ofthe invention for detecting a target sequence using a bipartite target probe having a sense promoter sequence for a double-stranded promoter and target-complementary sequences that are contiguous when annealed to a target nucleic acid having the target sequence.
  • FIG. 4 shows an embodiment of a method in which a circular transcription substrate is obtained by annealing a bipartite target probe having a sense promoter sequence for a double-sfranded promoter to a target sequence, ligating the annealed bipartite target probe, and then complexing the ligation product with an anti-sense promoter oligo.
  • the circular transcription substrate is amplified by rolling circle transcription.
  • FIG. 5 shows an embodiment of a method that uses coupled target-dependent rolling circle replication and run-off transcription of a linear transcription substrate that has a double-sfranded promoter to amplify the amount of transcription product obtained.
  • the copies of the target sequence in the rolling circle replication product are identical to the target sequence in the sample and provide additional sites for annealing and ligation of target probes in order to obtain more linear transcription substrates.
  • the embodiment shown here uses monopartite target probes to make a linear franscription substrate.
  • the invention also comprises embodiments that use a bipartite target probe to obtain a circular transcription substrate for rolling circle transcription.
  • FIG. 6 shows an embodiment of a method in which target-complementary sequences ofthe bipartite target probe are not contiguous when annealed to a target sequence and the gap between the target-complementary sequences is filled using a simple target probe.
  • FIG. 7 illustrates an embodiment of a method in which target-complementary sequences ofthe bipartite target probe are not contiguous when annealed to a target sequence and the gap between the target-complementary sequences is filled by DNA polymerase extension.
  • FIG. 8 shows an embodiment of the invention for detecting a target sequence by generating a linear transcription substrate using monopartite target probes, including a promoter target probe that comprises a sense promoter sequence for an RNA polymerase that uses a double-stranded promoter.
  • FIG. 9 shows a method to obtain additional amplification of transcription products.
  • FIG. 10 shows a method for detecting a non-nucleic acid analyte using an analyte-binding substance comprising an antibody that has a covalently- (e.g., chemically attached) or non-covalently- (e.g., using biotin and streptavidin) attached target sequence tag comprising a target sequence.
  • This example uses a bipartite target probe having a sense promoter sequence for a double-stranded promoter, such as a T7 RNAP promoter.
  • the circular franscription substrate has a transcription termination sequence so that multiple single RNA copies are obtained, rather than multimeric tandem copies of an oligomeric RNA, as obtained by rolling circle franscription.
  • FIG. 11 shows a method for detecting a non-nucleic acid analyte using an analyte-binding substance that has a target sequence tag comprising a target sequence, wherein the the signal for the analyte-binding substance and the analyte is generated by rolling circle franscription.
  • This example also uses a bipartite target probe having a sense promoter sequence for a double-stranded promoter, but does not have a franscription termination sequence so that multimeric tandem copies of an oligomeric RNA is obtained by rolling circle franscription.
  • FIG. 12 illustrates an example of different monopartite target probes for embodiments ofthe invention in which the promoter sequence comprises a sequence for an RNA polymerase that binds a single-sfranded promoter and initiates transcription therefrom.
  • the target probes are similar to embodiments that use an RNA polymerase that binds a double- stranded promoter except that embodiments that use single-stranded promoters are simpler since they do not use an anti-sense promoter oligo to make a franscription subsfrate ofthe invention.
  • the single-sfranded promoter sequence is a pseudopromoter for an RNA polymerase, such as but not limited to a pseudopromoter for a T7-type RNAP (e.g., T7 RNAP, T3 RNAP or SP6 RNAP), E. coli RNAP or a Thermus RNAP, and the cognate RNA polymerase for the promoter is used.
  • the single-sfranded promoter is an N4 promoter and the RNA polymerase is an N4 mini-vRNAP or a Y678F mutant of an N4 mini-vRNAP.
  • FIG. 13 shows an example of a bipartite target probe ofthe invention that comprises a single-sfranded promoter sequence for an RNA polymerase that uses a single- sfranded promoter for transcription.
  • the bipartite target probe is similar to a bipartite target probe that has a sense promoter sequence for a double-sfranded promoter except that a circular transcription substrate is obtained using a bipartite target probe that comprises a single-sfranded promoter without annealing of an anti-sense promoter oligo.
  • the single-sfranded promoter comprising a bipartite target probe is an N4 promoter and the RNA polymerase is an N4 mini-vRNAP or a Y678F mutant of an N4 mini-vRNAP.
  • the single- sfranded promoter is a pseudopromoter for an RNA polymerase, such as but not limited to a pseudopromoter for a T7-type RNAP (e.g., T7 RNAP, T3 RNAP or SP6 RNAP), E. coli RNAP or a Thermus RNAP, and the cognate RNA polymerase for the promoter is used.
  • the single-stranded promoter comprising a bipartite target probe is an N4 promoter and the RNA polymerase is an N4 mini-vRNAP or a Y678F mutant of an N4 mini-vRNAP.
  • the single- sfranded promoter is a pseudopromoter for an RNA polymerase, such as but not limited to a pseudopromoter for a T7-type RNAP (e.g., T7 RNAP, T3 RNAP or SP6 RNAP), E. coli RNAP or a Thermus RNAP, and the cognate RNA polymerase for the promoter is used.
  • RNA polymerase such as but not limited to a pseudopromoter for a T7-type RNAP (e.g., T7 RNAP, T3 RNAP or SP6 RNAP), E. coli RNAP or a Thermus RNAP, and the cognate RNA polymerase for the promoter is used.
  • FIG. 15 shows an embodiment of a method in which the circular franscription substrate obtained using a bipartite target probe having a single-sfranded promoter or pseudopromoter is amplified by rolling circle transcription.
  • the single-stranded promoter comprising a bipartite target probe is an N4 promoter and the RNA polymerase is an N4 mini-vRNAP or a Y678F mutant of an N4 mini-vRNAP.
  • the single- sfranded promoter is a pseudopromoter for an RNA polymerase, such as but not limited to a pseudopromoter for a T7-type RNAP (e.g., T7 RNAP, T3 RNAP or SP6 RNAP), E. coli RNAP or a Tliermus RNAP, and the cognate RNA polymerase for the promoter is used.
  • FIG. 16 shows an embodiment of a method that uses coupled target-dependent rolling circle replication and rolling circle transcription to amplify the amount of franscription product obtained.
  • IsoThermTM DNA Polymerase EPICENTRE Technologies, Madison, WI
  • Another strand-displacing DNA polymerase that can be used is RepliPHF M phi29 DNA polymerase (EPICENTRE Technologies, Madison, WI).
  • the copies ofthe target sequence in the rolling circle replication product are identical to the target sequence in the sample and provide additional sites for annealing and ligation of target probes in order to obtain more franscription substrates.
  • the embodiment shown here uses a bipartite target probe that comprises a single-stranded promoter or pseudopromoter to make a circular transcription substrate for rolling circle transcription by an RNA polymerase that binds the single-stranded promoter. Ligation ofthe bipartite target probe catenates the circular transcription substrate to the rolling circle replication product comprising the replicated target sequence. The catenated circular transcription substrates must be released from the rolling circle replication product to achieve efficient rolling circle transcription.
  • the method for releasing the catenated circular transcription substrates illustrated here is to include a quantity of dUTP in the rolling circle replication reaction mix in addition to dTTP so that a dUMP residue is incorporated randomly about every 100-400 nucleotides.
  • the single-sfranded promoter comprising a bipartite target probe is an N4 promoter and the RNA polymerase is an N4 mini-vRNAP or a Y678F mutant of an N4 mini- vRNAP.
  • the single-stranded promoter is a pseudopromoter for an RNA polymerase, such as but not limited to a pseudopromoter for a T7-type RNAP (e.g., T7 RNAP, T3 RNAP or SP6 RNAP), E. coli RNAP or a Tfiermus RNAP, and the cognate RNA polymerase for the promoter is used.
  • a pseudopromoter for a T7-type RNAP e.g., T7 RNAP, T3 RNAP or SP6 RNAP
  • E. coli RNAP e.g., E. coli RNAP or a Tfiermus RNAP
  • Tfiermus RNAP e.g., Tfiermus RNAP
  • FIG. 17 shows an embodiment of a method that uses coupled target-dependent rolling circle replication and run-off transcription of linear franscription substrates obtained by ligation of monopartite target probes annealed to the replicated target sequences in the rolling circle replication product to amplify the amount of transcription product.
  • the single-stranded promoter is a pseudopromoter for an RNA polymerase, such as but not limited to a pseudopromoter for a T7-type RNAP (e.g., T7 RNAP, T3 RNAP or SP6 RNAP), E.
  • RNAP coli RNAP or a Thermus RNAP, and the cognate RNA polymerase for the promoter is used, since these enzymes efficiently displace the RNA product from the DNA template strand.
  • an N4 mini-vRNAP enzyme can also be used together with a composition of EcoSSB Protein.
  • FIG. 18 shows an embodiment of a method in which the target-complementary sequences of a bipartite target probe that comprises a single-stranded promoter or pseudopromoter are not contiguous when annealed to a target sequence and the gap between the target-complementary sequences is filled using a simple target probe.
  • the circular transcription substrate has a transcription termination sequence so that only one copy ofthe transcription product is obtained, rather than a multimer of tandem oligomers as obtained from rolling circle transcription.
  • FIG. 19 illustrates an embodiment of a method in which the target- complementary sequences of a bipartite target probe that comprises a single-sfranded promoter or pseudopromoter are not contiguous when annealed to a target sequence and the gap between the target-complementary sequences is filled by DNA polymerase extension and subsequent ligation to obtain a circular transcription substrate.
  • FIG. 20 shows an embodiment ofthe invention for detecting a target sequence by generating a linear transcription substrate using monopartite target probes, wherein the promoter target probe comprises a single-sfranded promoter or pseudopromoter.
  • the single- stranded promoter is a pseudopromoter for a T7-type RNAP (e.g., T7 RNAP, T3 RNAP or SP6 RNAP) and the RNA polymerase is the cognate T7-type RNAP, or a pseudopromoter for E.
  • T7-type RNAP e.g., T7 RNAP, T3 RNAP or SP6 RNAP
  • the RNA polymerase is the cognate T7-type RNAP, or a pseudopromoter for E.
  • FIG. 21 shows a method to obtain additional amplification of transcription products.
  • the method uses two bipartite target probes comprising single-stranded promoters or pseudopromoters to generate circular franscription substrates for rolling circle franscription by a cognate RNA polymerase, and reverse transcription ofthe resulting RNA products to make additional copies of sense or anti-sense target sequences for annealing and ligation of additional first or second bipartite target probes, respectively, which in turn are used to transcribe more RNA, which is detected. [0075] FIG.
  • the signal for detection ofthe analyte-binding substance and the analyte is generated by transcription of a circular transcription substrate obtained by annealing and ligation of a bipartite target probe comprising a single- sfranded promoter or pseudopromoter.
  • the circular franscription subsfrate has a franscription termination sequence so that multiple single RNA copies are obtained, rather than multimeric tandem copies of an oligomeric RNA as obtained by rolling circle transcription.
  • FIG. 23 shows a method for detecting a non-nucleic acid analyte using an analyte-binding substance that has a target sequence tag comprising a target sequence, wherein the the signal for the analyte-binding substance and the analyte is generated by rolling circle transcription of a circular transcription subsfrate obtained by annealing and ligation of a bipartite target probe comprising a single-sfranded promoter or pseudopromoter.
  • the target sequence tag is designed to have a size so that a free 3 '-end is less than about 150 nucleotides and preferably less than 50-100 nucleotides from the target sequence, the catenated circular transcription substrates are easily released from the target sequence tag.
  • the analyte can be any of a broad range of analytes for which an analyte-binding substance is available or can be identified.
  • the single-stranded promoter comprising a bipartite target probe is an N4 promoter and the RNA polymerase is an N4 mini-vRNAP or a Y678F mutant of an N4 mini- vRNAP.
  • the single-sfranded promoter is a pseudopromoter for an RNA polymerase, such as but not limited to a pseudopromoter for a T7-type RNAP (e.g., T7 RNAP, T3 RNAP or SP6 RNAP), E. coli RNAP or a Thermus RNAP, and the cognate RNA polymerase for the promoter is used.
  • a pseudopromoter for a T7-type RNAP e.g., T7 RNAP, T3 RNAP or SP6 RNAP
  • E. coli RNAP e.g., E. coli RNAP or a Thermus RNAP
  • the cognate RNA polymerase for the promoter is used.
  • the present invention discloses novel methods, processes, compositions, and kits for amplifying and detecting one or multiple target nucleic acid sequences in or from a sample, including target sequences that differ by as little as one nucleotide.
  • the target sequence or target sequences can comprise at least a portion of one or more target nucleic acids comprising either RNA or DNA from any source, or a target sequence can comprise a target sequence tag that is attached to an analyte-binding substance, such as, but not limited to, an antibody, thus permitting use ofthe methods and compositions ofthe present invention to detect any analyte for which there is a suitable analyte-binding substance.
  • the methods ofthe invention involve obtaining franscription products using a transcription subsfrate as a template, wherein the transcription subsfrate is made by ligating at least two different target-complementary sequences comprising one or more target probes when the target-complementary sequences are annealed adjacently on the target sequence. Since the target sequence is required for annealing and ligation ofthe target-complementary sequences which makes the franscription substrate, obtaining the transcription product is target-dependent. Therefore, detection ofthe transcription products is indicative ofthe presence ofthe target sequence comprising the target nucleic acid or the target sequence tag joined to an analyte-binding substance in the sample.
  • the present invention comprises novel methods, compositions and kits for amplifying, detecting and quantifying one or multiple target nucleic acid sequences in a sample, including target sequences that differ by as little as one nucleotide.
  • the target sequence or target sequences can comprise one or more target nucleic acids comprising either RNA or DNA from any source.
  • the methods can also be used to detect an analyte of any type for which an analyte-binding substance (such as, but not limited to, an antibody) can be obtained, provided that a tag comprising a target nucleic acid sequence is coupled or linked to said analyte-binding substance.
  • the method is useful for detecting specific nucleic acids or analytes in a sample with high specificity and sensitivity.
  • the method also has an inherently low level of background signal.
  • Prefe ⁇ ed embodiments ofthe method consist of an annealing process, a DNA ligation process, an optional DNA polymerase extension process, a transcription process, and, optionally, a detection process.
  • the DNA ligation joins a probe which has a first target- complementary sequence and a sense promoter sequence for an RNA polymerase to another probe or another section ofthe same probe which has a second target-complementary sequence and, optionally, a signal sequence.
  • This step is dependent on hybridization ofthe target- complementary probe sequences to a target sequence and forms a ligation product which, upon complexing with an anti-sense promoter oligo, makes a transcription substrate for in vitro transcription ofthe second target-complementary sequence and the signal sequence, if a signal sequence is present, in an amount that is proportional to the amount of target sequence in the sample.
  • RNA polymerase preferably a T7-type RNA polymerase, and most preferably, T7 RNA polymerase, and synthesizes a franscription product using the transcription promoter and a single-sfranded DNA template which is operably or functionally joined or linked to the promoter using a method of the invention.
  • Joining ofthe sense promoter sequence to the franscription template, which yields a franscription substrate upon complexing with an anti-sense promoter sequence is target- dependent because joining by ligation only occurs if the different target-complementary sequences comprising the target probes are adjacent to or abut each when they anneal to a target sequence, if the target sequence is present in the sample.
  • the methods ofthe invention are therefore referred to herein as "target-dependent transcription.”
  • the amount of franscription product obtained in a given reaction time can also be increased using a coupled rolling circle replication and target-dependent transcription reaction.
  • the rolling circle replication reaction uses a "target sequence amplification probe” (or a “TSA probe") having target-complementary sequences at each end and an intervening sequence with a primer binding site.
  • the TSA probe anneals to the target sequence, if present in the sample, and is ligated to form a "TSA circle.”
  • TSA circle After annealing a primer to the primer binding site, rolling circle replication ofthe TSA circle by a strand-displacing DNA polymerase under strand- displacing polymerization conditions generates multiple tandem copies ofthe target sequence, which serve as annealing and ligation sites for one or more target probes ofthe invention.
  • Ligation joins a sense promoter sequence and a first target-complementary sequence to one or more other target-complementary sequences.
  • a transcription substrate is obtained for an RNA polymerase that binds the double-sfranded promoter and synthesizes transcription products comprising multiple copies ofthe target sequence.
  • FIG. 9 Yet another method for obtaining additional amplification ofthe target sequence is illustrated schematically in Figure 9. This method generates annealing and ligation sites for a second bipartite target probe by reverse transcription ofthe transcription products obtained following annealing of a first bipartite target probe to a target sequence in the sample, ligation of the bipartite target probe, and in vitro transcription ofthe resulting circular transcription subsfrate.
  • RNA complementary to target-complementary probe sequences can be detected and quantified using any ofthe conventional detection systems for nucleic acids such as detection of fluorescent labels, enzyme-linked detection systems, antibody-mediated label detection, and detection of radioactive labels.
  • the signal sequence in the transcription substrate can comprise a sequence that is amplifiable and/or detectable by another method.
  • a signal sequence that encodes a substrate for an enzyme such as, but not limited to Q-beta replicase is used.
  • in vitro franscription ofthe ssDNA transcription subsfrate results in synthesis of a subsfrate for a replicase, which is used to rapidly and linearly amplify the signal further. Since the amplified product is directly proportional to the amount of target sequence present, quantitative measurements reliably represent the amount of a target sequence in a sample.
  • Major advantages of this method are that the ligation process, or an optional DNA polymerase extension, can be manipulated to obtain single-nucleotide allelic discrimination, the transcription process is isothermal, and signals are strictly quantitative because the transcription reaction is linear and is catalyzed by a highly processive enzyme, and signal amplification can be obtained which is also linear and greatly enhances the sensitivity of an assay or method. In multiplex assays, the transcription promoter sequence used for in vitro transcription can be the same for all target probes.
  • a "nucleic acid” or “polynucleotide” or “oligonucleotide” (or “oligo") ofthe invention is a polymer molecule comprising a series of “mononucleosides,” also referred to as “nucleosides,” in which the 3 '-position ofthe pentose sugar of one nucleoside is linked by an internucleoside linkage, such as, but not limited to, a phosphodiester bond, to the 5 '-position of the pentose sugar ofthe next nucleoside.
  • a nucleoside linked to a phosphate group is referred to as a "nucleotide.”
  • the nucleotide that is linked to the 5'-position ofthe next nucleotide in the series is referred to as "5 '-of,” or “upsfream of,” or the “5 '-nucleotide” and the nucleotide that is linked to the 3 '-position of said 5' or upstream nucleotide is referred to as "3 '-of,” or “downstream of,” or the "3'-nucleotide.”
  • the former may be called the "upstream" polynucleotide or oligonucleotide
  • a "portion" or “region,” used interchangeably herein, of a polynucleotide or oligonucleotide is a contiguous sequence of 2 or more bases.
  • a region or portion is at least about any of 3-5, 5-10, 10-15, 15-20, 20-25, 25-50, 50-100, 100-200, 200-400, 400-600, 600-800, 800-1000, 1000-1500, or greater than 1500 contiguous nucleotides.
  • a portion or region can be 5 '-of or 3 '-of another portion or genetic element or sequence.
  • a portion or region can also comprise a 5'-end portion or a 3'-end portion, meaning it comprises a 5 '-end or a 3 '-end, respectively, or it can be a portion or region that is between a 5'- portion and a 3 '-portion.
  • a circular oligonucleotide or polynucleotide does not have an end or an end portion, it can have portions or regions that are 5 '-of or 3 '-of another portion or region or sequence or genetic element, which permits orientation of one portion or region or sequence or genetic element with respect to another within the circular nucleic acid sfrand.
  • nucleic acid chemistry and structure particularly related to the 3'- and 5 '-positions of sugar moieties of canonical nucleic acid nucleotides, and in the context of enzymatic synthesis of nucleic acids in a 5'-to-3' direction. Since most descriptions of embodiments ofthe present invention are referring to single-stranded nucleic acids, in most cases herein the inventors use the terms "3 '-of ' and "5 '-of to refer to the relative position or orientation of a particular nucleic acid sequence or genetic element encoded by a sequence that is located on the same nucleic acid strand.
  • a transcription promoter that is "3'-of the target sequence” refers to the position of a promoter relative to a target sequence on the same strand.
  • the pentose sugar of the nucleic acid can be ribose, in which case, the nucleic acid or polynucleotide is referred to as "RNA,” or it can be 2'-deoxyribose, in which case, the nucleic acid or polynucleotide is refe ⁇ ed to as "DNA.”
  • the nucleic acid can be composed of both DNA and RNA mononucleotides. In both RNA and DNA, each pentose sugar is covalently linked to one of four common "nucleic acid bases" (each also referred to as a "base").
  • oligonucleotide is defined as a molecule comprised of two or more deoxyribonucleotides or ribonucleotides, preferably about 10 to 200 nucleotides, but there is no defined limit to the length of an oligonucleotide.
  • a nucleic acid or polynucleotide ofthe invention may comprise one or more modified nucleic acid bases, sugar moieties, or internucleoside linkages.
  • nucleic acids or polynucleotides that contain modified bases, sugar moieties, or internucleoside linkages include, but are not limited to: (1) modification ofthe T m ; (2) changing the susceptibility ofthe polynucleotide to one or more nucleases; (3) providing a moiety for attachment of a label; (4) providing a label or a quencher for a label; or (5) providing a moiety, such as biotin, for attaching to another molecule which is in solution or bound to a surface.
  • the invention does not limit the composition ofthe nucleic acids or polynucleotides ofthe invention including any target probes, detection probes, such as, but not limited to molecular beacons (U.S. Patents Nos. 5,925,517 and 6,103,476 of Tyagi et al. and U.S. Patents No. 6,461,817 of Alland et al., all of which are incorporated herein by reference); capture probes, oligonucleotides, or other nucleic acids used or detected in the assays or methods, so long as each said nucleic acid functions for its intended use.
  • molecular beacons U.S. Patents Nos. 5,925,517 and 6,103,476 of Tyagi et al. and U.S. Patents No. 6,461,817 of Alland et al., all of which are incorporated herein by reference
  • the nucleic acid bases in the mononucleotides may comprise guanine, adenine, uracil, thymine, or cytidine, or alternatively, one or more ofthe nucleic acid bases may comprise xanthine, allyamino-uracil, hypoxanthine, 2-aminoadenine, 6- methyl and other alkyl adenines, 2-propyl and other alkyl adenines, 5-halouracil, 5-halo cytosine, 5-propynyl uracil, 5-propynyl cytosine, 7-deazaadenine, 7-deazaguanine, 7-deaza-7- methyl-adenine, 7-deaza-7-methyl-guanine, 7-deaza-7-propynyl-adenine, 7-deaza-7-propynyl-adenine and other 7-deaza-7-alkyl or 7-aryl purines,
  • nucleic acid base that is derivatized with a biotin moiety, a digoxigenin moiety, a fluorescent or chemiluminescent moiety, a quenching moiety or some other moiety.
  • the invention is not limited to the nucleic acid bases listed; this list is given to show the broad range of bases which may be used for a particular purpose in a method. [0092] When a molecule comprising both a nucleic acid and a peptide nucleic acid
  • modified bases can be used in one or both parts.
  • binding affinity can be increased by the use of certain modified bases in both the nucleotide subunits that make up the 2'-deoxyoligonucleotides ofthe invention and in the peptide nucleic acid subunits.
  • modified bases may include 5-propynylpyrimidines, 6-azapyrimidines, and N-2, N-6 and 0-6 substituted purines including 2-aminopropyladenine.
  • Other modified pyrimidine and purine base are also expected to increase the binding affinity of macromolecules to a complementary sfrand of nucleic acid.
  • one or more of the sugar moieties can comprise ribose or 2'-deoxyribose, or alternatively, one or more ofthe sugar moieties can be some other sugar moiety, such as, but not limited to, 2'-fluoro-2'- deoxyribose or 2'-0-methyl-ribose, which provide resistance to some nucleases.
  • the internucleoside linkages of nucleic acids or polynucleotides ofthe invention can be phosphodiester linkages, or alternatively, one or more ofthe internucleoside linkages can comprise modified linkages, such as, but not limited to, phosphorothioate, phosphorodithioate, phosphoroselenate, or phosphorodiselenate linkages, which are resistant to some nucleases.
  • modified linkages such as, but not limited to, phosphorothioate, phosphorodithioate, phosphoroselenate, or phosphorodiselenate linkages, which are resistant to some nucleases.
  • a variety of methods are known in the art for making nucleic acids having a particular sequence or that contain particular nucleic acid bases, sugars, internucleoside linkages, chemical moieties, and other compositions and characteristics.
  • any one or any combination of these methods can be used to make a nucleic acid, polynucleotide, or oligonucleotide for the present invention.
  • the methods include, but are not limited to: (1) chemical synthesis (usually, but not always, using a nucleic acid synthesizer instrument); (2) post-synthesis chemical modification or derivatization; (3) cloning of a naturally occurring or synthetic nucleic acid in a nucleic acid cloning vector (e.g., see Sambrook, et al., Molecular Cloning: A Laboratory Approach Second Edition, 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY and Sambrook and Russell, Molecular Cloning, A Laboratory Manual, Third Edition, 2001, Cold Spring Harbor Laboratory Press,) such as, but not limited to a plasmid, bacteriophage (e.g., Ml 3 or lamba), phagemid, cosmid, fosmid, YAC, or BAC cloning vector, including vectors for producing single-sfran
  • coli RNA polymerase or SP6 or T7 R&DNATM Polymerase (EPICENTRE Technologies, Madison, WI, USA), or another enzyme; (8) use of restriction enzymes and/or modifying enzymes, including, but not limited to exo- or endonucleases, kinases, ligases, phosphatases, methylases, glycosylases, terminal fransferases, including kits containing such modifying enzymes and other reagents for making particular modifications in nucleic acids; (9) use of polynucleotide phosphorylases to make new randomized nucleic acids; (10) other compositions, such as, but not limited to, aribozyme ligase to join RNA molecules; and/or (11) any combination of any ofthe above or other techniques known in the art.
  • restriction enzymes and/or modifying enzymes including, but not limited to exo- or endonucleases, kinases, ligases, phosphatases, methylases, glycosy
  • Oligonucleotides and polynucleotides including chimeric (i.e., composite) molecules and oligonucleotides with modified bases, sugars, or internucleoside linkages are commercially available (e.g., TriLink Biotechnologies, San Diego, CA, USA or Integrated DNA Technologies, Coralville, IA).
  • hybridize or “anneal” and “hybridization” or “annealing” refer to the formation of complexes between nucleotide sequences on opposite or complementary nucleic acid strands that are sufficiently complementary to form complexes via Watson-Crick base pairing.
  • a target probe, primer, transcription substrate, or another oligonucleotide or polynucleotide "hybridizes” or “anneals” with target nucleic acid or a template or another oligonucleotide or polynucleotide
  • such complexes or “hybrids” are sufficiently stable to serve the function required for ligation, DNA polymerase extension, or other function for which it is intended.
  • a "template” is a nucleic acid molecule that is being copied by a nucleic acid polymerase.
  • the synthesized copy is complementary to the template.
  • Both RNA and DNA are always synthesized in the 5'-to-3' direction and the two strands of a nucleic acid duplex always are aligned so that the 5 '-ends ofthe two strands are at opposite ends ofthe duplex (and, by necessity, so then are the 3 '-ends).
  • DNA polymerases including both DNA-dependent (i.e, having a DNA template) and RNA-dependent (i.e., having an RNA template, which enzyme is also called a "reverse transcriptase" DNA polymerases, require a primer for synthesis of DNA.
  • RNA polymerases do not require a primer for RNA synthesis.
  • a "template” or a “ligation template” or a “template for ligation” is a nucleic acid molecule to which two or more complementary oligonucleotides, target probes, or other nucleic acids that are to be ligated anneal or hybridize prior to ligation, wherein the ends of said nucleic acid molecules that are to be ligated are adjacent to each other when annealed to the ligation template.
  • sample or a “biological sample” according to the present invention is used in its broadest sense.
  • a sample is any specimen that is collected from or is associated with a biological or environmental source, or which comprises or contains biological material, whether in whole or in part, and whether living or dead.
  • a sample can also be chemically synthesized or derived in the laboratory, rather than from a natural source.
  • Biological samples may be plant or animal, including human, fluid (e.g., blood or blood fractions, urine, saliva, sputum, cerebral spinal fluid, pleural fluid, milk, lymph, or semen), swabs (e.g., buccal or cervical swabs), solid (e.g., stool), microbial cultures (e.g., plate or liquid cultures of bacteria, fungi, parasites, protozoans, or viruses), or cells or tissue (e.g., fresh or paraffin-embedded tissue sections, hair follicles, mouse tail snips, leaves, or parts of human, animal, plant, microbial, viral, or other cells, tissues, organs or whole organisms, including subcellular fractions or cell extracts), as well as liquid and solid food and feed products and ingredients such as dairy items, vegetables, meat and meat by-products, and waste.
  • fluid e.g., blood or blood fractions, urine, saliva, sputum, cerebral spinal fluid, pleural fluid, milk, lymph,
  • Biological samples may be obtained from all ofthe various families of domestic plants or animals, as well as wild animals or plants.
  • Environmental samples include environmental material such as surface matter, soil, water, air, or industrial samples, as well as samples obtained from food and dairy processing instruments, apparatus, equipment, utensils, disposable and non-disposable items. These examples are not to be construed as limiting the sample types applicable to the present invention.
  • a sample comprises a specimen from any source that contains or may contain a target nucleic acid.
  • a sample on which the assay method ofthe invention is carried out can be a raw specimen of biological material, such as serum or other body fluid, tissue culture medium or food material. More typically, the method is carried out on a sample that is a processed specimen, derived from a raw specimen by various treatments to remove materials that would interfere with detection of a target nucleic acid or an amplification product thereof. Methods for processing raw samples to obtain a sample more suitable for the assay methods ofthe invention are well known in the art.
  • an "analyte” means a substance or a part of a substance whose presence, concentration or amount in a sample is being determined in an assay.
  • An analyte is sometimes referred to as a "target substance” or a “target molecule” or a “target analyte” of an assay.
  • An analyte may also be refe ⁇ ed to more specifically.
  • the present invention pertains to analytes that are target nucleic acid sequences that comprise or are in a "target nucleic acid” or a “target polynucleotide” or a “target oligonucleotide.”
  • a composition, kit, or method ofthe invention can be used for an "analyte-specif ⁇ c reagent" to detect an analyte comprising a target nucleic acid sequence in a sample.
  • an analyte is often associated with a biological entity that is present in a sample if and only if the analyte is present.
  • biological entities include viroids (analyte is, e.g., a segment of a viroid nucleic acid sequence); viruses (analyte is, e.g., a sequence in the viral genome); other microorganisms (analyte is, e.g., a sequence in the genome or the RNA ofthe microorganism); abnormal cells, such as cancer cells (analyte is, e.g., a sequence in an oncogene); or an abnormal gene (analyte is, e.g., a sequence in a gene segment that includes the altered bases which render the gene abnormal or in a messenger RNA segment that includes altered bases as a result of having been transcribed from the abnormal gene).
  • viroids analyte is, e.g., a segment of a viroid nucle
  • an analyte can be chemically synthesized sequence or derived in the laboratory for a particular purpose, rather than from a natural source.
  • the analyte can be a chemically synthesized oligonucleotide tag that comprises a target sequence that is covalently or non- covalently attached to an analyte-binding substance such as an antibody in order to indirectly detect another analyte in the sample which is bound by the analyte-binding substance.
  • the oligonucleotide tag that is attached or joined to an analyte-binding substance can be referred to as a "target sequence” or a “target sequence tag,” even though it is used to detect and/or quantify a protein, lipid, carbohydrate or another analyte by detecting the analyte-binding substance to which the target sequence is joined.
  • the present invention has widespread applicability, including in applications in which nucleic acid probe hybridization assays or immunoassays are often employed.
  • the invention is useful in diagnosing diseases in plants and animals, including humans; and in testing products, such as food, blood, and tissue cultures, for contaminants.
  • a "target” of the present invention is a biological organism or material that is the reason or basis for which a diagnostic assay is performed.
  • an assay ofthe present invention may be performed to detect a target that is a virus which is indicative of a present disease or a risk of future disease (e.g., H wh ch is believed to result in AIDS), or a target that is a gene which is indicative of antibiotic resistance (e.g., an antibiotic resistance gene in an infectious pathogenic bacterium), or a target that is a gene which, if absent, maybe indicative of disease (e.g., a deletion in an essential gene).
  • a target analyte that is a sequence in a "target polynucleotide” or a “target nucleic acid” comprises at least one nucleic acid molecule or portion of at least one nucleic acid molecule, whether said molecule or molecules is or are DNA, RNA, or both DNA and RNA, and wherein each said molecule has, at least in part, a defined nucleotide sequence.
  • the target polynucleotide may also have at least partial complementarity with other molecules which can be used in an assay, such as, but not limited to, capture probes.
  • a capture probe for this purpose is complementary to a different region of a target nucleic acid than the target sequence and may have a moiety, such as a biotin moiety, that permits immobilization of the target nucleic acid on a surface, such as a surface to which streptavidin is attached.
  • the target polynucleotide may be single- or double- stranded.
  • a target sequence ofthe present invention may be of any length. However, it must comprise a sequence of sufficient sequence specificity and length so as to be useful for its intended purpose.
  • a target sequence that is to be detected using target sequence- complementary target probes must have a sequence of sufficient sequence specificity and length so as remain hybridized by said target probes under assay hybridization conditions wherein sequences that are not target sequences are not hybridized.
  • a target sequence in a target polynucleotide having sufficient sequence specificity and length for an assay ofthe present invention may be identified, using methods known to those skilled in the art, by comparison and analysis of nucleic acid sequences known for a target and for other sequences which may be present in the sample. For example, sequences for nucleic acids of many viruses, bacteria, humans (e.g., for genes and messenger RNA), and many other biological organisms can be searched using public or private databases, and sequence comparisons, folded structures, and hybridization melting temperatures (i.e., T m 's) may be obtained using computer software known to those knowledgeable in the art.
  • a method of the present invention can be carried out on nucleic acid from a variety of sources, including unpurified nucleic acids, or nucleic acids purified using any appropriate method in the art, such as, but not limited to, various "spin” columns, cationic membranes and filters, or salt precipitation techniques, for which a wide variety of products are commercially available (e.g., MasterPureTM DNA & RNA Purification Kits from EPICENTRE Technologies, Madison, WI, USA). Methods ofthe present invention can also be carried out on nucleic acids isolated from viroids, viruses or cells of a specimen and deposited onto solid supports as described by Gillespie and Spiegelman (J. Mol. Biol.
  • the method can also be carried out with nucleic acid isolated from specimens and deposited on solid support by "dot” blotting (Kafatos, et al., Nucl. Acids Res., 7: 1541-1552, 1979); White, and Bancroft, J. Biol. Chem., 257: 8569-8572, 1982); Southern blotting (Southern, E., J. Mol. Biol., 98: 503-517, 1975); “northern” blotting (Thomas, Proc. Natl. Acad. Sci.
  • the method can also be carried out for nucleic acids spotted on membranes, on slides, or on chips as arrays or microarrays. Nucleic acid of specimens can also be assayed by the method ofthe present invention applied to water phase hybridization (Britten, and Kohne, Science, 161: 527-540, 1968) and water/organic interphase hybridizations (Kohne, et al., Biochemistry, 16: 5329-5341, 1977).
  • Water/organic interphase hybridizations have the advantage of proceeding with very rapid kinetics but are not suitable when an organic phase-soluble linking moiety, such as biotin, is joined to the nucleic acid affinity molecule.
  • the methods ofthe present invention can also be carried out on amplification products obtained by amplification of a naturally occurring target nucleic acid, provided that the target sequence in the target nucleic acid is amplified by the method used only if the target nucleic acid is present in the sample. Suitable amplification methods include, but are not limited to, PCR, RT-PCR, NASBA, TMA, 3SR, LCR, LLA, SDA (e.g., Walker et al., Nucleic Acids Res.
  • nucleic acid that is a product of another amplification method as a target nucleic acid for an assay ofthe present invention, such as, but not limited to, for obtaining more sensitive detection of targets, greater specificity, or to decrease the time required to obtain an assay result.
  • Nucleic acid used as a template for amplification is isolated from cells contained in the biological sample, according to standard methodologies (Sambrook et al., In: Molecular Cloning: A Laboratory Manual 2 rev.ed., Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 1989).
  • the nucleic acid may be genomic DNA or fractionated or whole cell RNA. Where RNA is used, it may be desired to convert the RNA to a complementary DNA. In one embodiment, the RNA is whole cell RNA and is used directly as the template for amplification. >
  • primers that selectively hybridize to nucleic acids are contacted with the isolated nucleic acid under conditions that permit selective hybridization.
  • the term "primer,” as defined herein, is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process.
  • primers are oligonucleotides from ten to twenty or thirty base pairs in length, but longer sequences can be employed.
  • Primers may be provided in double-sfranded or single-stranded form, although the single-sfranded form is preferred.
  • the nucleic acid:primer complex is contacted with one or more enzymes that facilitate template-dependent nucleic acid synthesis. Multiple rounds of amplification, also referred to as “cycles,” are conducted until a sufficient amount of amplification product is produced.
  • the amplification product is detected.
  • the detection may be performed by visual means.
  • the detection may involve indirect identification ofthe product via chemiluminescence, radioactive scintigraphy of incorporated radiolabel or fluorescent label, such as real-time analysis with SYBR® Green dye, or even via a system using electrical or thermal impulse signals (Affymax technology).
  • a number of template dependent processes are available to amplify the marker sequences present in a given template sample.
  • One ofthe best known amplification methods is the polymerase chain reaction (referred to as PCR) which is described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159.
  • a reverse transcriptase PCR amplification procedure may be performed in order to quantify the amount of mRNA amplified.
  • Alternative methods for reverse transcription utilize thermostable, RNA-dependent DNA polymerases. These methods are described in WO 90/07641, filed Dec.21, 1990. Polymerase chain reaction methodologies are well known in the art.
  • the methods of the invention can also be carried out on nucleic acids isolated from specimens and deposited onto solid supports by dot-blotting, or by adso ⁇ tion onto walls of microtiter plate wells or solid support materials on dipsticks, on membranes, on slides, or on chips as arrays or microarrays.
  • the amplified target-complementary sequences of target probes ofthe invention can also be hybridized to oligonucleotides or nucleic acids attached to or deposited on slides, chips or other surfaces, such as, but not limited to arrays or microarrays, for detection and identification.
  • the methods ofthe invention are applicable to detecting target sequences in cellular nucleic acids in whole cells, including single cells, from a specimen, such as a fixed or paraffin-embedded section, or from microorganisms immobilized on a solid support, such as replica-plated bacteria or yeast.
  • the methods ofthe invention can be used to amplify and/or detect target nucleic acid sequences in living cells.
  • the invention is also not limited to detection of analytes comprising a target nucleic acid.
  • the present invention provides assays, methods, compositions and kits for detection and quantification of an analyte of any type in a sample.
  • Target Sequences Comprising Target Nucleic Acids in a Sample or a Target Sequence Tag Joined to an Analyte-Binding Substance
  • target nucleic acid sequence refers to the particular nucleotide sequence ofthe target nucleic acid(s) that is/are to be detected.
  • a “target sequence” comprises one or more sequences within one or more target nucleic acids, which target nucleic acid can be naturally occurring in a sample or a target sequence tag that is joined or attached to an analyte-binding substance.
  • the target nucleic acid may be either single-stranded or double-sfranded and may include other sequences besides the target sequence.
  • a target nucleic acid is sometimes referred to more specifically by the type of nucleic acid.
  • a target nucleic acid can be a "target RNA” or an "RNA target,” or a "target mRNA,” or a "target DNA” or a “DNA target.”
  • the target sequence can be referred to as "a target RNA sequence” or an "RNA target sequence”, or as a "target mRNA sequence” or a “target DNA sequence,” or the like.
  • the target sequence comprises one or more entire target nucleic acids.
  • the target sequence comprises only a portion of one or more nucleic acid molecules.
  • target sequence sometimes is also used to refer to the particular target-complementary nucleotide sequences that is/are present in the target-complementary "target probes” used in a method or assay ofthe invention, but more preferably, these sequences are referred to as "target-complementary sequences.”
  • target sequence refers only to that portion ofthe sequence of a target nucleic acid for which a complementary sequence is present in a target probe ofthe invention.
  • multiple target probes are used, including target probe sets that are complementary to other target sequences in a target nucleic acid, which other target sequences can be on the same or the opposite nucleic acid strand ofthe same target nucleic acid, or on another target nucleic acid in the sample or joined to another analyte-binding substance.
  • a transcription promoter is present in a target probe that is complementary to only one strand ofthe target sequence, and in other embodiments, a transcription promoter is present in two different target probes - one that is complementary to a target sequence on one strand, and the other that is complementary to a complementary target sequence on the other strand, wherein the transcription promoters can be the same or different in each case.
  • the target sequence in a method or assay ofthe invention will be a known sequence or one of a small number of known sequences, such as, but not limited to one or a small number of sequences comprising known specific mutations or single nucleotide polymo ⁇ hisms (SNPs), or one of known sequences that are specific for and identify a particular organism or group of closely-related organisms.
  • a known sequence or one of a small number of known sequences such as, but not limited to one or a small number of sequences comprising known specific mutations or single nucleotide polymo ⁇ hisms (SNPs), or one of known sequences that are specific for and identify a particular organism or group of closely-related organisms.
  • target nucleotide is part of a target sequence and comprises the nucleotide position that differs between "wild-type” or “normal” alleles and single-base “mutant” alleles, or the nucleotide that differs between different "wild-type” alleles that comprise different single-nucleotide polymo ⁇ hisms (SNPs) for a particular nucleotide position in a target nucleic acid.
  • a target nucleic acid ofthe present invention comprising a target sequence to be detected and/or quantified includes nucleic acids from any source in purified or unpurified form.
  • target nucleic acids can be any DNA, including, but not limited to, dsDNA and ssDNA, such as mitochondrial DNA, chloroplast DNA, chromosomes, plasmids or other episomes, the genomes of bacteria, yeasts, viruses, viroids, mycoplasma, molds, or other microorganisms, or genomes of fungi, plants, animals, or humans, or target nucleic acids can be any RNA, including, but not limited to, tRNA, mRNA, rRNA, mitochondrial RNA, chloroplast RNA, or target nucleic acids can be mixtures of DNA and RNA, including, but not limited to, mixtures ofthe above nucleic acids or fragments thereof or DNA-RNA hybrids.
  • the target nucleic acid can be only a minor fraction of a complex mixture such as a biological sample and can be obtained from various biological materials by procedures known in the art. As discussed herein above, methods for purification of a target nucleic, if further purification is necessary, are also known in the art.
  • An initial step prior to amplification of a target nucleic acid sequence is rendering the target nucleic acid single-sfranded.
  • the target nucleic acid is a double-stranded DNA (dsDNA)
  • the initial step is target denaturation.
  • the denaturation step may be thermal denaturation or any other method known in the art, such as alkali treatment.
  • the ssDNA target sequence comprises either ssDNA that is present in a biological sample or ssDNA that is obtained by denaturation of dsDNA in the sample.
  • the ssDNA target sequence comprises ssDNA that is obtained as a result of a "primer extension reaction,” meaning an in vitro or in vivo DNA polymerization reaction using either ssDNA or denatured dsDNA that is present in the sample as a template and an oligonucleotide as a primer under DNA polymerization reaction conditions.
  • the target nucleic acid in the sample or the primer extension product, or both are made into smaller DNA fragments by methods known in the art in order to generate a DNA target sequence for use in the methods ofthe invention.
  • the initial step may be the synthesis of a single- sfranded cDNA.
  • Techniques for the synthesis of cDNA from RNA are known in the art.
  • the ssDNA target sequence comprises first-strand cDNA obtained by reverse franscription ofthe RNA target, meaning an in vitro reaction that utilizes an RNA present in a sample as a template and a nucleic acid oligonucleotide that is complementary to at least a portion of a sequence ofthe RNA template as a primer in order to synthesize ssDNA using an RNA-dependent DNA polymerase (i.e., reverse transcriptase) under reaction conditions.
  • RNA-dependent DNA polymerase i.e., reverse transcriptase
  • a first-sfrand cDNA for use in methods ofthe invention is synthesized in situ in cells or tissue in a tissue section using methods such as those described in U.S. Patent Nos. 5,168,038; 5,021,335; and 5,514,545, which are inco ⁇ orated herein by reference.
  • Target Probes of the Invention Simple Target Probes; Promoter Target Probes; Signal Target Probes; Monpartite Target Probes; Bipartite Target Probes
  • a “target probe” of the present invention is a linear single-stranded oligonucleotide that comprises at least one sequence that is "a target-complementary sequence,” meaning a sequence that is complementary to a portion of a target sequence comprising a target nucleic acid or a target sequence tag, and wherein the target probe is used in an assay or method ofthe invention.
  • target probes comprise deoxyribonucleotides having canonical nucleic acid bases and internucleoside linkages, although modified sugars, bases or internucleoside linkages can be used for a particular pu ⁇ ose as discussed elsewhere herein.
  • the size and nucleotide composition of a target-complementary sequence of a target probe can vary.
  • the target-complementary sequences of all target probes ofthe invention must be of sufficient length and nucleotide composition so as to anneal with specificity to a complementary target sequence with which it is perfectly based-paired under the conditions used in the assay or method for annealing of target probes to the target sequence and for ligation ofthe target probes that are annealed to the target sequence, under which conditions, target probes that are not complementary to the target sequence do not remain annealed and, if not perfectly basepaired at the ligation junction, do not ligate.
  • the length of a target-complementary sequence can vary based in part on its sequence and on the T m of that sequence, and on the temperature and other reaction conditions that are used for annealing ofthe target probes and ligation ofthe target probes on the target sequence.
  • a target-complementary sequence of a target probe will comprise at least four nucleotides if the target-complementary sequences are annealed to the target sequence and ligated at a temperature that is less than or equal to about 30° C, or at least about eight nucleotides if the target-complementary sequences are annealed to the target sequence and ligated at a temperature that is greater than about 30° C.
  • a target- complementary sequence of only 4-8 nucleotides is not sufficient to provide the desired nucleotide specificity in an assay or method ofthe invention.
  • a target-complementary sequence of a target probe that is complementary to the 5'-end or to the 3'-end ofthe target sequence comprises about 10 to about 100 nucleotides, and most preferably, about 15 to about 50 nucleotides.
  • those with knowledge in the art will know how to empirically determine the optimal lengths of target-complementary sequences for target probes for particular target sequences, and under the particular conditions, which conditions can vary with respect to factors such as but not limited to temperature, ionic strength, concentration of co-solvents such as but not limited to betaine, or other factors.
  • the length of one target-complementary sequence of a bipartite target probe can be different than the other.
  • the length of a target-complementary sequence of one monopartite target probe can be different than the lengths of target-complementary sequences of other monopartite target probes used in the assay or method.
  • the sequence of a target probe that is complementary to the 3'-end of a target sequence is designed to be longer than the sequence of a target probe that is joined to a sense promoter sequence and that is complementary to the 5'-end ofthe target sequence, although the sequence of a target probe that is complementary to the 3 '-end ofthe target sequence need not be longer, and can be about the same size or even shorter than the sequence that is complementary to the 5'-end ofthe target sequence.
  • the hybridization complex between that target probe and the target sequence will be more stable so that the ability to form a ligation junction will be more dependent on the annealing ofthe target- complementary sequence that is joined to the sense promoter sequence and that anneals to the 5'- end ofthe target sequence.
  • the length ofthe target-complementary sequence that is joined to the sense promoter sequence comprises a sufficient number of nucleotides so as anneal to the 5'-end ofthe target sequence with specificity under the conditions ofthe assay or method, but is optimized so that transcription of said target-complementary sequence is minimized unless and until it is ligated to another target-complementary sequence that is adjacently annealed on the target sequence.
  • the optimal length ofthe target- complementary sequence that is joined to the sense promoter sequence can vary for different RNA polymerases.
  • the optimal length of a target-complementary sequence that is joined to a sense T7 RNAP promoter sequence will be the shortest sequence that anneals to the target sequence with specificity. This appears to be due to the fact that T7 RNAP, which has RNA:DNA hybrid unwinding activity, displaces both long and very short transcripts from the template and binding to the promoter and initiation of transcription appear to be the rate limiting steps for transcription. On the other hand, N4 mini- vRNAP does not have RNA:DNA hybrid unwinding activity and EcoSSB Protein appears to be responsible for displacing the RNA transcript from the template strand.
  • a target probe comprises an N4 vRNAP promoter
  • the length ofthe target-complementary sequence that is joined to the N4 promoter sequence is designed, based on the information of Davidova et al., so that a transcript made by an N4 mini-vRNAP will either not be displaced or at least, EcoSSB-activated displacement is minimized from the target- complementary sequence unless and until this sequence is ligated to an adjacent target- complementary sequence that is annealed to the target sequence to make a transcription subsfrate ofthe invention.
  • the simple target probe(s) that is/are used to fill the gap can be of any length so long as they provide suitable ligation junctions for joining to the target-complementary sequences that are annealed to the 3'- and 5'-ends ofthe target sequence.
  • target probe of the invention which is called a "simple target probe,” comprises a linear single-sfranded oligonucleotide comprising only a sequence that is complementary to one continuous portion of a target sequence.
  • a “promoter target probe” comprises a 5 '-end portion that is complementary to the most 5'-portion of a target sequence.
  • the 3'-end ofthe target-complementary portion is joined to the 5 '-end of a sense promoter sequence which, upon complexing with an anti-sense promoter sequence, serves as a functional transcription promoter for an RNA polymerase that synthesizes RNA under transcription conditions using said franscription promoter and ssDNA that is 5 '-of said promoter (with respect to the same sfrand) as a template.
  • a promoter target probe can also comprise other "optional sequences" that do not comprise a target- complementary sequence or a promoter sequence, which optional sequences, if present, are 3 '-of said promoter sequence in said promoter target probe.
  • Such optional sequences in said promoter target probe can serve other functions in a method or assay ofthe invention.
  • a “signal target probe” comprises a 3 '-portion and a 5 '-portion, wherein, the 3 '-end portion of said signal probe comprises a sequence that is complementary to the most 3 '-portion of a target sequence, and said 5 '-portion comprises a "signal sequence,” wherein said signal sequence comprises a sequence that is detectable in some way, directly or indirectly, following transcription of said signal sequence that is joined, in the presence of a target sequence, to a target-complementary sequence and a sense promoter sequence during an assay or method ofthe present invention.
  • a signal target probe can also comprise other "optional sequences" that do not comprise the signal sequence, which sequences can serve another function in a method or assay ofthe invention, or they can have no function, other than to connect the signal sequence to one or more other sequences.
  • "Monopartite target probes" of the present invention are target probes that comprise only one sequence that is complementary to one portion of a target sequence.
  • the target-complementary sequence in a monopartite target probe is not interrupted by any other sequence that is not complementary to the target sequence.
  • Promoter target probes and signal target probes are monopartite target probes that are used to generate linear franscription substrates in some embodiments of assays and methods ofthe invention.
  • Simple target probes are monopartite target probes that can be used in embodiments ofthe invention to generate either linear transcription substrates or circular franscription substrates, as discussed herein below.
  • Simple target probes are monopartite target probes that are used in embodiments ofthe invention in order to fill at least a portion of a gap region between target-complementary sequences of other target probes that are not contiguous when annealed to a target sequence.
  • one or more simple target probes can be used in methods and assays ofthe invention that generate a linear franscription subsfrate by annealing to a target sequence between the sequences ofthe target sequence to which the target-complementary sequences of a promoter target probe and a signal target probe anneal.
  • Figure 1 illustrates monopartite target probes and shows one embodiment of how monopartite target probes are oriented when annealed to a target sequence.
  • the target-complementary sequences of the promoter target probe and the signal target probe are contiguous or adjacent when they are annealed to a target sequence and a simple target probe is not used.
  • the target-complementary sequences ofthe promoter target probe and the signal target probe are not contiguous when they are annealed to a target sequence, but rather than using a simple target probe to anneal to the gap region on the target sequence between the the target-complementary sequences ofthe promoter target probe and the signal target probe, the gap is "filled" by DNA polymerase extension from the 3 '-end ofthe signal target probe.
  • One or more simple target probes can also be used in embodiments of methods and assays ofthe invention that generate a circular franscription subsfrate, in which case, the simple target probe(s) anneal to a target sequence between the regions ofthe target sequence to which the target-complementary sequences at the ends of a bipartite target probe anneal.
  • the simple target probe(s) anneal to a target sequence between the regions ofthe target sequence to which the target-complementary sequences at the ends of a bipartite target probe anneal.
  • other embodiments ofthe invention which are preferred embodiments, use a bipartite target probe and generate a circular transcription subsfrate.
  • a “bipartite target probe” is refe ⁇ ed to as “bipartite” because it comprises two different target-complementary sequences, each of which is complementary to a different portion of a target sequence, which target- complementary sequences are separated within the bipartite target probe by other sequences that are not complementary to the target sequence.
  • the target-complementary sequences in a bipartite target probe are in two parts or "bipartite.”
  • a bipartite target probe comprises a ssDNA that has a 5 '-end that resembles a promoter target probe and a 3 '-end that resembles either a simple target probe or a signal target probe.
  • the 5'-end of a bipartite target probe has a sequence that is complementary to the most 5 '-portion of a target sequence and, then on the same strand, 3 '-ofthe target-complementary sequence, a sense transcription promoter sequence which, upon complexing with an anti-sense promoter sequence, can bind an RNA polymerase that can make a franscription product under transcription conditions using single-sfranded DNA that is joined 5 '-of said promoter as a template.
  • the 3 '-end of a bipartite target probe comprises a sequence that is complementary to the most 3 '-end portion of said target sequence.
  • a signal sequence can be 5 '-of the target-complementary sequence at the 3 '-end of a bipartite target probe, although a signal sequence does not need to be contiguous with or immediately adjacent to the target-complementary sequence at the 3 '-end of a bipartite target probe.
  • Other sequences that can have other functions or that have no function other than to join the two sequences can be between a target-complementary sequence at the 3 '-end and a signal sequence of a bipartite target probe.
  • the target-complementary sequences of a bipartite target probe need not be contiguous or immediately adjacent when they are annealed to a target sequence.
  • bipartite target-complementary sequences are not contiguous or immediately adjacent when they are annealed to a target sequence, then one or more simple target probes that are complementary to the portions ofthe target sequence between the target-complementary sequences ofthe bipartite target probe can be used in some embodiments of methods or assays ofthe invention.
  • a DNA polymerase can be used to "fill in” where there is no target probe annealed to the target sequence by primer-extending from the 3'-hydroxyl end of a bipartite target probe that is annealed to a target sequence using the target sequence as a template.
  • Figure 2 illustrates a bipartite target probe ofthe invention and shows how different sequence portions of a bipartite target probe are oriented with respect to each other when said bipartite target probe is free in solution and when it is annealed to a target sequence.
  • the embodiment illustrated in Figure 2 shows a bipartite target probe comprising target- complementary sequences at each end that are contiguous or adjacent when annealed to a target sequence.
  • the invention also comprises other embodiments of bipartite target probes wherein the target-complementary sequences at each end that are not contiguous or adjacent when annealed to a target sequence.
  • the "gap" between the target-complementary sequences ofthe bipartite target probe can be "filled" using one or more simple target probes or by DNA polymerase extension from the 3 '-end ofthe bipartite target probe using the target sequence as a template.
  • all target probes ofthe invention including both monopartite and bipartite target probes that are joined with a ligase in a method or assay ofthe invention, will have a phosphate group at their 5'-end and a hydroxyl group at their 3'-end.
  • the 5'-ends of target probes that are not joined with a ligase to the 3 '-end of another target probe in a method or assay ofthe invention do not have a phosphate group on their 5'-ends.
  • Another joining method such as, but not limited to a chemical joining method or a topoisomerase-mediated joining method.
  • secondary or additional amplification of a target sequence and/or a signal sequence is obtained by using a "second target probe," which second target probe can comprise either: (i) "second monopartite target probes” comprising a "second promoter target probe” and either a "second signal target probe,” if a signal sequence is present, or a "second simple target probe,” and one or more additional “second simple target probes; or (ii) a "second bipartite target probe.” If a second target probe is used, then the target probes that are complementary to the target sequence are referred to as "first target probes.”
  • a second target probe is generally identical to a first target probe except with respect to the target- complementary sequence of said target probe.
  • sequence at the 5'-end of a second promoter target probe or at the 5'-end of a second bipartite target probe is complementary to the target-complementary sequence at the 3'-end ofthe first signal target probe or to the target-complementary sequence at the 3'-end ofthe bipartite target probe, respectively.
  • sequence at the 3'-end of a second signal target probe or at the 3 '-end of a second bipartite target probe is complementary to the target-complementary sequence at the 5'-end ofthe first promoter target probe or to the target-complementary sequence at the 5'- end ofthe bipartite target probe, respectively.
  • a second simple target probe is complementary to a first target probe.
  • a second target probe can also be referred to as an "target amplification probe,” which can comprise either: (i) “monopartite target amplification probes” comprising a “promoter target amplification probe” and either a “signal target amplification probe,” if a signal sequence is present, or a “simple target amplification probe,” and one or more additional “simple target amplification probes; or (ii) a "bipartite target amplification probe.”
  • the target probes used in said assay or method are designed in order to be able to distinguish said target nucleotide(s).
  • the nucleotide of a target probe ofthe invention that is complementary to the target nucleotide comprises either the 5 '-end of a promoter target probe if the assay or method generates a linear transcription subsfrate, or the nucleotide at the 5' -end of a bipartite target probe if the assay or method generates a cirular franscription subsfrate.
  • the complementary nucleotide at the 5 '-end ofthe respective promoter target probe or the bipartite target probe will anneal thereto and will be ligated, respectively, either to the 3 '-end of an adjacently-annealed monopartite target probe or to an adjacently-annealed 3'-end ofthe bipartite target probe.
  • the 5'-end ofthe promoter target probe or the 5'-end ofthe bipartite target probe is not complementary to the target nucleotide, it will not anneal thereto, and said 5 '-end will not be ligated to the 3 '-end of an adjacently-annealed monopartite target probe or the 3 '-end ofthe bipartite target probe, respectively, during the ligation process.
  • the non-complementarity ofthe 5 '-end of a target probe with a target nucleotide in a target sequence when the target probes of an assay or method are annealed to the target sequence prevents ligation of said 5 '-end with a 3'-hydroxyl end, so that a franscription substrate is not formed.
  • the preferred nucleotide of a target probe ofthe invention that is complementary to the target nucleotide comprises either the 5'-end of a promoter target probe if the assay or method generates a linear transcription substrate, or the nucleotide at the 5 '-end of a bipartite target probe if the assay or method generates a cirular transcription substrate
  • the invention also comprises other embodiments of target probes in which the nucleotide that is complementary to the target nucleotide comprises a different nucleotide in a monopartite or bipartite target probe.
  • the nucleotide that is complementary to the target nucleotide can comprise either the 3 '-end ofthe signal target probe if a signal sequence is present, or the 3 '-end ofthe simple target probe if a signal sequence is not used.
  • nucleotide that is complementary to the target nucleotide can comprise the 3 '-end of said bipartite target probe.
  • nucleotide that is complementary to the target nucleotide can comprise a nucleotide at either the 3 '-end or the 5 '-end of one of said simple target probes that is vised to fill the gap.
  • the nucleotide that is complementary to the target nucleotide comprises a nucleotide at either the 3 '-end or the 5 '-end of a simple target probe used to fill the gap that anneals to the target sequence adjacent to the promoter target probe, and most preferably, the nucleotide that is complementary to the target nucleotide comprises a nucleotide at the 3 '-end of said simple target probe.
  • the nucleotide that is complementary to the target nucleotide can comprise a nucleotide at either the 3'-end or the 5'-end of one of said simple target probes that is used to fill the gap.
  • the nucleotide that is complementary to the target nucleotide comprises a nucleotide at either the 3 '-end or the 5 '-end of a simple target probes used to fill the gap that anneals to the target sequence adjacent to the 5 '-end of said bipartite target probe, and most preferably, the nucleotide that is complementary to the target nucleotide comprises a nucleotide at the 3 '-end of said simple target probe.
  • one or more 5'-terminal or 3'-terminal nucleotide positions of a target probe used in an assay or method to detect a particular target nucleotide in a target sequence may not comprise a sequence that will anneal with specificity to said target sequence, such as, when said target nucleotide is part of a target sequence that has a low T m with respect to said target-complementary sequence of said target probe.
  • a method or assay ofthe invention does not need to use a signal sequence.
  • the use of a signal sequence or a signal target probe in a method or assay ofthe invention is optional. If a signal sequence is used in an embodiment, the invention is not limited with respect to particular signal sequences that can be used in signal target probes or bipartite target probes of the invention.
  • a signal sequence can comprise any sequence that generates a detectable signal or that enables sensitive and specific detection, whether directly or indirectly, ofthe generation of an RNA transcription product encoded by the signal sequence.
  • a signal sequence encodes an RNA product that results in additional amplification or more sensitive detection.
  • one signal sequence that can be used in a signal target probe or a bipartite target probe ofthe present invention is a sequence that encodes a substrate for a replicase, such as, but not limited to, Q-beta replicase or a partial or interrupted sequence for a subsfrate for a replicase, such as, but not limited to, Q-beta replicase.
  • Q-beta replicase substrates and methods that can be used for making and using signal sequences that encode a partial or interrupted replicase substrate for signal target probes are described in U.S. Patent No. 6,562,575, inco ⁇ orated herein by reference.
  • a complete sequence for a replicase subsfrate is preferred in a signal probe ofthe present invention, but a sequence of a partial or interrupted replicase substrate is used in embodiments that require reduced background signal (or "noise") and greater sensitivity. If the time for appearance of a signal in an assay or method is shortened by amplifying the amount of transcription product using other methods described herein, it is less likely that a sequence for a partial or interrupted replicase substrate, rather than for a complete replicase substrate, is needed to obtain a good signal to noise ratio in the assay or method.
  • RNA that is a substrate for Q-beta replicase is synthesized, incubation of said RNA substrate with Q-beta replicase results in replication ofthe substrate, thereby resulting in additional amplification ofthe signal and more sensitive, though indirect, detection ofthe presence of a target sequence.
  • a “replicase” according to the invention is an enzyme that catalyzes exponential synthesis (i.e., "replication") of an RNA substrate.
  • the replicase can be from any source for which a suitable exponentially replicatable substrate can be obtained for use in the invention.
  • the replicase is an RNA-directed RNA polymerase.
  • the replicase is a bacteriophage replicase, such as Q-beta replicase, MS2 replicase, or SP replicase.
  • the replicase is Q-beta replicase.
  • the replicase is isolated from eucaryotic cells infected with a virus, such as, but not limited to, cells infected with brome mosaic virus, cowpea mosaic virus, cucumber mosaic virus, or polio virus.
  • the replicase is a DNA-directed RNA polymerase, in which case, a T7-like RNA polymerase (as defined in U.S. Pat. No.4,952,496) is preferred, and T7 RNA polymerase (Konarska, M. M., and Sha ⁇ , P. A., Cell, 63: 609-618, 1990) is most preferred.
  • the replicase can be prepared from cells containing a virus or from cells expressing a gene from a bacteriophage or a eukaryotic virus cloned into a plasmid or other vector.
  • replication of a Q-beta replicase substrate can be carried out substantially according to the protocol of Kramer et al. (J. Mol. Biol., 89: 719-736, 1974). Briefly, an RNA substrate is incubated at 37° C.
  • one ofthe NTPs can be labeled with a fluorescent or other dye, or the replication products can be detected using another method, such as but not limited to by detection of fluorescence that results from intercalation of a dye such as ethidium bromide.
  • the sequence of the recombinant subsfrate or template be derived from the sequence of an RNA in the following group: midivariant RNA (MDV-1 RNA), microvariant RNA, nanovariant RNA, CT RNA, RQ135 RNA, RQ120 RNA, and other variants or Q-beta RNA, which are known in the art.
  • Another signal sequence that can be used is an expressable gene for an enzyme that has a substrate that results in a colored or fluorescent or otherwise detectable product.
  • a gene for a green fluorescent protein (GFP) can be used.
  • GFP green fluorescent protein
  • in vitro transcription of a transcription substrate generated by target-dependent annealing and ligation of target probes results in an RNA transcript that encodes a GFP.
  • a detectable GFP is synthesized.
  • a signal sequence can comprise a binding site for another molecule, such as, but not limited to, a molecular beacon that results in a signal.
  • a molecular beacon that results in a signal.
  • a monopartite or a bipartite target probe of the present invention can optionally comprise other "optional sequences."
  • Optional sequences if present, can be 5'-of the target- complementary sequence at the 3 '-end and 3 '-of the promoter sequence in the 5 '-portion of a bipartite target probe.
  • Optional sequences, if present in a monopartite target probe can be 5 '-of the target-complementary sequence in a signal target probe or 3 '-of the promoter sequence in a promoter target probe.
  • other optional sequences can comprise one or more transcription termination sequences, one or more capture sequence sites, one or more detection sequence sites, one or more address tag sites, one or more priming sites, one or more sequences for another specific purpose, or one or more intervening sequences that have no function other than to link one portion of a target probe to another portion.
  • a capture sequence site can be a site that is complementary to another oligonucleotide, such as, but not limited to an oligo with a biotin group, that facilitates capture of a target sequence to a surface, such as a surface to which streptavidin is bound.
  • a detection sequence site can be a sequence that is complementary to an oligo used for detection, such as, but not limited to, a molecular beacon.
  • An address tag can be a sequence that is complementary to an oligonucleotide or a polynucleotide that is attached to a surface, such as, but not limited to, a dipstick or a spot on an array or microarray.
  • a priming site can be for a sequence that is complementary to an oligonucleotide primer, such as, but not limited to a primer for use in reverse franscription of an RNA transcript product of an assay or method ofthe invention.
  • a "transcription substrate” ofthe present invention means a polynucleotide that comprises a target-complementary sequence that is operably joined to a functional promoter for an RNA polymerase that can make a transcription product using the target-complementary sequence as a template under transcription conditions.
  • a transcription substrate is a polynucleotide complex that results from covalent joining in the presence of a target sequence of at least two target-complementary sequences comprising at least two monopartite target probes or at least one bipartite target probe, wherein the 3 '-end ofthe target-complementary sequence that anneals to the 5 '-end ofthe target sequence is joined to a sense promoter sequence that is complexed with (or annealed to) an anti-sense promoter oligo to obtain a double-sfranded promoter, wherein an RNA polymerase can bind said double-sfranded promoter and initiate transcription therefrom under transcription conditions to obtain a franscription product.
  • a transcription substrate ofthe invention can also have additional nucleic acid sequences, such as but not limited to detectable "signal sequences," that are in the same DNA sfrand and 5 '-of said joined target-complementary sequences.
  • a franscription substrate ofthe invention is not required to have said additional nucleic acid sequences.
  • a transcription subsfrate comprises a target sequence that is operably joined to a single-stranded promoter or pseudopromoter for an RNA polymerase that can bind said single-sfranded promoter or pseudopromoter and initiate transcription therefrom.
  • a transcription subsfrate typically has a franscription initiation site at the 5 '-end ofthe promoter sequence.
  • a franscription subsfrate ofthe invention can also have one or more other sequences that are 5 '-of the target-complementary sequence.
  • a franscription substrate can have one or more franscription termination sequences, one or more sites for DNA cleavage to permit controlled linearization of a circular franscription subsfrate, and/or other sequences or genetic elements for a particular pu ⁇ ose, including, but not limited to, sequences that are transcribed by the RNA polymerase so as to provide additional regions of complementarity in the RNA transcription products: (i) for annealing of primers for reverse transcription in order to make cDNA for additional rounds of amplification; or (ii) for annealing of additional target probes for generation of additional transcription substrates by means of additional joining reactions using the RNA franscription product as a ligation template (e.g., by using a different joining enzyme or joining method on the RNA ligation template than the joining enzyme or joining method that was used in the
  • Hybridization or “annealing” refers to the “binding” or “pairing” of complementary nucleic acid bases in one single-stranded nucleic acid, peptide nucleic acid (PNA), or linked nucleic acid-PNA molecule with another single-sfranded nucleic acid, PNA, or linked nucleic acid-PNA molecule under "binding” or “annealing” or “hybridization” conditions.”
  • PNA peptide nucleic acid
  • PNA peptide nucleic acid
  • linked nucleic acid-PNA molecule with another single-sfranded nucleic acid, PNA, or linked nucleic acid-PNA molecule under “binding” or “annealing” or “hybridization” conditions.
  • Hybridization occurs according to base pairing rules (e.g., adenine pairs with thymine or uracil and guanine pairs with cytosine).
  • base pairing rules e.g., adenine pairs with thymine or uracil and guanine pairs with cytosine.
  • an assay ofthe invention in developing and making binding conditions for particular target nucleic acid analytes or target sequence tags joined to non-nucleic acid analytes with target probes an assay ofthe invention, as well as in developing and making hybridization conditions for other oligonucleotides or polynucleotides which can be used, such as, but not limited to capture probes, or detection probes such as molecular beacons, certain additives can be added in the hybridization solution.
  • dextran sulfate or polyethylene glycol can be added to accelerate the rate of hybridization (e.g., Chapter 9, Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2 nd Edition, Cold Spring Harbor Laboratory Press, 1989), or betaine can be added to the hybridization solution to eliminate the dependence of T m . on basepair composition (Rees, W.A., et al., Biochemistry, 32, 137-144, 1993).
  • other hybridization conditions that do not use such additives can also be used in an assay or method ofthe invention.
  • degree of homology or “degree of complementarity” refer to the extent or frequency at which the nucleic acid bases on one sfrand (e.g., ofthe affinity molecule) are “complementary with” or “able to pair” with the nucleic acid bases on the other sfrand (e.g., the analyte).
  • Complementarity may be "partial,” meaning only some ofthe nucleic acid bases are matched according to base pairing rules, or complementarity maybe “complete” or “total.”
  • the length i.e., the number of nucleic acid bases comprising the nucleic acid and or PNA affinity molecule and the nucleic acid analyte
  • the degree of "homology” or “complementarity” between the affinity molecule and the analyte have significant effects on the efficiency and strength of binding or hybridization when the nucleic acid bases on the affinity molecule are maximally “bound” or “hybridized” to the nucleic acid bases on the analyte.
  • the terms “melting temperature” or “T m " are used as an indication ofthe degree of complementarity.
  • the T m is the temperature at which a population of double-sfranded nucleic acid molecules becomes half dissociated into single strands under defined conditions. Based on the assumption that a nucleic acid molecule that is used in hybridization will be approximately completely homologous or complementary to a target polynucleotide, equations have been developed for estimating the T m for a given single-sfranded sequence that is hybridized or "annealed" to a complementary sequence.
  • T m 81.5° C +0.41(%G+C) when the nucleic acid is in an aqueous solution containing 1 M NaCl (see e.g., Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid Hybridization, 1985).
  • Other more sophisticated equations available for nucleic acids take nearest neighbor and other structural effects into account for calculation ofthe T m . Binding is generally stronger for PNA affinity molecules than for nucleic acid affinity molecules.
  • the T m of a 10-mer homothymidine PNA binding to its complementary 10-mer homoadenosine DNA is 73° C
  • the T m for the corresponding 10-mer homothymidine DNA to the same complementary 10-mer homoadenosine DNA is only 23° C.
  • Equations for calculating the T m for a nucleic acid are not appropriate for PNA.
  • a T m that is calculated using an equation in the art is checked empirically and the hybridization or binding conditions are adjusted by empirically raising or lowering the stringency of hybridization as appropriate for a particular assay.
  • stringency is used in reference to the conditions of temperature, ionic strength, and the presence of other compounds, under which nucleic acid hybridizations are conducted. With “high stringency” conditions, nucleic acid base pairing will occur only between nucleic acid fragments that have a high frequency of complementary base sequences. Thus, conditions of "weak” or “low” stringency are often required when it is desired that nucleic acids that are not completely complementary to one another be hybridized or annealed together.
  • hybridization As used herein, “hybridization,” “hybridizes” or “capable of hybridizing” is understood to mean the forming of a double or triple sfranded molecule or a molecule with partial double or triple sfranded nature.
  • the term “hybridization,” “hybridize(s)” or “capable of hybridizing” encompasses the terms “stringent condition(s)” or “high stringency” and the terms “low stringency” or “low stringency condition(s).”
  • stringent conditions or “high stringency conditions” comprise conditions that allow hybridization between or within one or more nucleic acid strands containing complementary sequences, but preclude hybridization of random sequences. Stringent conditions tolerate little, if any, mismatch between a nucleic acid and a target sfrand. Such conditions are well known to those of ordinary skill in the art, and are preferred for applications requiring high selectivity. Non-limiting applications include isolating a nucleic acid, such as a gene or a nucleic acid segment thereof, or detecting at least one specific mRNA transcript or a nucleic acid segment thereof, and the like.
  • Stringent conditions may comprise low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCl at temperatures of about 50° C to about 70° C. It is understood that the temperature and ionic strength of a desired stringency are determined in part by the length ofthe particular nucleic acid(s), the length and nucleobase content ofthe target sequence(s), the charge composition ofthe nucleic acid(s), and to the presence or concentration of formamide, tetramethyl-ammonium chloride, betaine or other solvent(s) in a hybridization mixture.
  • a medium stringency condition could be provided by about 0.1 to 0.25 M NaCl at temperatures of about 37° C to about 55° C. Under these conditions, hybridization may occur even though the sequences of probe and target sfrand are not perfectly complementary, but are mismatched at one or more positions.
  • a low stringency condition could be provided by about 0.15 M to about 0.9 M salt, at temperatures ranging from about 20° C to about 55° C.
  • hybridization may be achieved under conditions of 50 mM Tris-HCl (pH 8.3), 75 mM KC1, 3 mM MgCl , 1.0 mM dithiothreitol, at temperatures between approximately 20° C to about 37° C.
  • Other hybridization conditions utilized could include approximately 10 mM Tris-HCI (pH 8.3), 50 mM KC1, 1.5 mM MgCl 2 , at temperatures ranging from approximately 40° C to about 72° C.
  • complementarity it is important for some assays ofthe invention to determine whether the hybridization represents complete or partial complementarity. For example, where it is desired to detect simply the presence or absence of pathogen DNA (such as from a virus, bacterium, fungi, mycoplasma, protozoan), it is only important that the hybridization method ensures hybridization when the relevant sequence is present. In those embodiments ofthe invention, conditions can be selected where both partially complementary probes and completely complementary probes will hybridize. However, in general, even if the probes are only partially complementary, they must be completely complementary at the terminal nucleotides comprising the ligation junction.
  • pathogen DNA such as from a virus, bacterium, fungi, mycoplasma, protozoan
  • the invention can also be used for assays to detect mutations, or genetic polymo ⁇ hisms, or single nucleotide polymo ⁇ hisms (SNPs). These embodiments ofthe invention require that the hybridization and other aspects ofthe method distinguish between partial and complete complementarity.
  • human hemoglobin is composed, in part, of four polypeptide chains. Two of these chains are identical chains of 141 amino acids (alpha chains) and two of these chains are identical chains of 146 amino acids (beta chains).
  • the gene encoding the beta chain is known to exhibit polymo ⁇ hism.
  • the normal allele encodes a beta chain having glutamic acid at the sixth position.
  • the mutant allele encodes a beta chain having valine at the sixth position.
  • ligation refers to the joining of a 5 '-phosphorylated end of one nucleic acid molecule with the 3'-hydroxyl end of another nucleic acid molecule by an enzyme called a "ligase," although in some methods ofthe invention, the ligation can be effected by another mechanism. With respect to ligation, a region, portion, or sequence that is “adjacent to” or “contiguous to” or “contiguous with” another sequence directly abuts that region, portion, or sequence.
  • the invention is not limited to a specific ligase.
  • the ligase is not active in ligating blunt ends and is highly selective for ligation of a deoxyribonucleotide having a 5 '-phosphate and a deoxyribonucleotide having 3'-hydroxyl group when these respective 5'- and 3'- nucleotides are adjacent to each other when annealed to a target sequence of a target nucleic acid.
  • Ampligase® Thermostable DNA Ligase Tth DNA ligase, and Tfl DNA Ligase (EPICENTRE Technologies, Madison, Wisconsin, USA), or Tsc DNA Ligase (Prokaria Ltd., Reykjavik, Iceland) are NAD-dependent thermostable ligases that are not active on blunt ends and that ligate the 5 '-phosphate and 3'-hydroxyl termini of DNA ends that are adjacent to one another when annealed to a complementary DNA molecule; these enzymes are preferred ligases in embodiments ofthe invention wherein a target sequence comprises DNA.
  • Another DNA ligase that can be used in the methods ofthe invention for target sequences comprising DNA is Pfu DNA ligase as described by Mathur et al.
  • Thermostable DNA ligases are preferred in some embodiments because they can be cycled through multiple annealing-ligation-melting cycles, permitting multiple target probe ligations for every target sequence present in a sample, and thus, increasing the sensitivity of the assay or method.
  • the invention is not limited to the use of a particular ligase, or to the use of a thermostable ligase and other suitable ligases that function in the assays and methods o the invention can also be used.
  • T4 DNA ligase can be used in some embodiments ofthe invention for target sequences that comprise DNA.
  • T4 RNA ligase can efficiently ligate DNA ends of nucleic acids that are adjacent to each other when hybridized to an RNA sfrand.
  • T4 RNA ligase is a preferred ligase ofthe invention in embodiments in which DNA ends are ligated on a target sequence that comprises RNA.
  • T4 RNA ligase is not preferred when high specificity and/or high sensitivity is desired.
  • ligases that ligate DNA ends of nucleic acids that are adjacent to each other when hybridized to an RNA sfrand are preferred for target nucleic acids comprising RNA.
  • the invention is also not limited to the use of a ligase for covalently joining target probe ends in the various embodiments ofthe invention.
  • other ligation methods such as, but not limited to, topoisomerase-mediated ligation (e.g., U.S. Patent No. 5,766,891, inco ⁇ orated herein by reference) can be used, although topoisomerase-mediated ligation is not preferred in most embodiments because ofthe high potential for background ligation.
  • chemical ligation methods can be used, such as, but not limited to, the use of a target probe with a 5'-end sequence that comprises a 5'-iodo-nucleotide and a 3'-end comprising a nucleotide with phosphorothioate, as disclosed by Xu, Y., and Kool, E.T. (Nucleic Acids Res., 27: 875-881, 1999), which is inco ⁇ orated herein by reference.
  • ligation refers to any suitable method for joining adjacent 5'- and 3 '-ends of target probes that are adjacent or contiguous to each other when annealed to a target sequence.
  • all ofthe target probes that anneal to a target sequence have a similar melting temperature (T m ) with respect to the target sequence, and the lowest temperature at which a ligation process is performed is near the T m ofthe target probe having the lowest T m when it is annealed to the target sequence.
  • T m melting temperature
  • the present invention comprises some embodiments in which a TSA circle is replicated by rolling circle replication while catenated to target nucleic acid or a target sequence tag using a DNA polymerase, such as but not limited to IsoThermTM DNA polymerase (EPICENTRE Technologies, Madison, WI), Bst DNA polymerase large fragment, or another DNA polymerase that can efficiently replicate catenated templates.
  • a DNA polymerase such as but not limited to IsoThermTM DNA polymerase (EPICENTRE Technologies, Madison, WI), Bst DNA polymerase large fragment, or another DNA polymerase that can efficiently replicate catenated templates.
  • the present invention also comprises embodiments in which circular franscription substrates are transcribed by rolling circle franscription while they are catenated to a target nucleic acid or target sequence tag so long as the assay or method functions for its intended pu ⁇ ose. It can be beneficial that the respective molecules remain catenated to the target nucleic acid, and it is beneficial if the number of steps and the
  • circular ssDNA ligation products obtained using a method of the invention does not remain catenated to a target nucleic acid or target sequence tag comprising the target sequence following ligation.
  • catenation of a TSA circle obtained by annealing and ligation of a target sequence amplification probe (TSA probe) on a target sequence or catenation of a circular transcription substrate obtained by annealing and ligation of a bipartite target probe on a target sequence and annealing of an anti-sense promoter oligo may limit the amount of rolling circle replication product (e.g., see Baner, J.
  • Whether or not it is necessary to release catenated circular ssDNA molecules from the target nucleic acid prior to rolling circle replication depends on the DNA polymerase used, indicating that the need to release catenated circular transcription substrates from the target nucleic acid may also depend on the particular RNA polymerase used for rolling circle transcription.
  • the present invention comprises empirically determining if catenation of a ligation product obtained from ligation of a TSA probe or a bipartite target probe on the target sequence results in a reduction in the amount of product obtained during rolling circle replication or rolling circle franscription, respectively, compared to the amount of product obtained on an oligodeoxyribonucleotide comprising only the target sequence.
  • an assay or method ofthe invention will either use additional steps to release the catenated circular ssDNA molecule from the target nucleic acid for the particular assay or method, such as but not limited to one ofthe methods to release catenated molecules described herein below, or will use a different polymerase for which the amount of replication product or transcription product is not affected by catenation.
  • the circular ligation product is annealed to an anti-sense promoter oligo that is attached to a solid support in order to obtain a circular franscription substrate and to separate the circular transcription substrate from other nucleic acids and other components ofthe sample prior to in vitro transcription.
  • the target sequence comprising a target nucleic acid or a target sequence tag that is joined to an analyte-binding substance is less than about 150-200 nucleotides from the 3'-end ofthe respective target nucleic acid or target sequence tag.
  • a target sequence tag ofthe present invention will comprise a sequence that has a 3 '-end that is less than about 150-200 nucleotides from the target sequence.
  • the 3'-end ofthe target sequence tag is less than 100 nucleotides from the target sequence and most preferably, the 3'-end ofthe target sequence tag is less than 50 nucleotides from the target sequence.
  • target sequence comprising a target nucleic acid in a sample
  • the target sequence is more than about 150-200 nucleotides from the 3'-end ofthe target nucleic acid
  • any suitable method can be used to obtain a target nucleic acid for an assay or method ofthe present invention.
  • the target nucleic acid is fragmented to a size that has a 3 '-end that is less than about 150-200 nucleotides from the target sequence prior to use ofthe target nucleic acid in an assay or method ofthe invention.
  • a DNA in a sample comprising a dsDNA molecule or a ssDNA molecule to which an appropriate complementary DNA oligo is annealed can be digested with a restriction endonuclease, provided that a suitable restriction site is present within less than about 150-200 nucleotides from the 3'-end ofthe target sequence and no restriction sites for the enzyme are present within the target sequence.
  • one or more DNA oligonucleotides having a double-sfranded segment that contains a Fokl restriction enzyme site and a single-sfranded segment that binds to the desired cleavage site on a first-strand cDNA can be used.
  • this type of oligonucleotide can be used with the restriction enzyme Fokl to cut a single-stranded DNA at almost any desired sequence (Szybalski, W., Gene 40:169-173, 1985; Podhajska A. J. and Szybalski W., Gene 40:175, 1985, inco ⁇ orated herein by reference).
  • RNA or DNA nucleic acids of known sequence can be cleaved at specific sites using a 5 '-nuclease or CleavaseTM enzyme and specific oligonucleotides, as described by Kwiatkowski, et al., (Molecular Diagnosis 4:353-364, 1999) and in U.S. Patent No. 6,001,567 and related patents assigned to Third Wave Technologies (Madison, WI, USA), which are inco ⁇ orated herein by reference.
  • target nucleic acid is first-sfrand cDNA obtained by reverse transcription of
  • the RNA can be cleaved with RNase H at a site to which a DNA oligo is annealed in order to define the 3'-end ofthe reverse transcription product that is obtained.
  • the length ofthe reverse transcription product can be kept within a desired size range by limiting the time ofthe reverse franscription reaction, which reverse transcription reaction can be optimized for the particular primer, template sequence and reaction conditions used to obtain a target nucleic acid comprising a target sequence, if present in the sample.
  • Still another method that can be used is to inco ⁇ orate dUMP randomly into the first-strand cDNA during reverse franscription or primer extension to prepare a target nucleic acid comprising a target sequence.
  • dUTP deoxyribouridine triphosphate
  • dTTP thymidine triphosphate
  • dUTP can be inco ⁇ orated in place of a portion of the dTTP in rolling circle replication of TSA circles that are used to increase the number of target sequences available for annealing and ligation of target probes for target-dependent transcription.
  • TSA circles are obtained by annealing and ligation of target sequence amplification probes (TSA probes) on a target sequence in a sample.
  • dUMP When dUTP is used in a reverse franscription, primer extension or rolling circle replication reaction in addition to dTTP, dUMP will be inco ⁇ orated randomly in place of IMP at a frequency based on the ratio of dUTP to dTTP. Then, the respective first- strand cDNA, primer extension product or rolling circle replication product can be cleaved at sites of dUMP inco ⁇ oration by treatment (e.g., see U.S. Patent No. 6,048,696, inco ⁇ orated herein by reference) with uracil-N-glycosylase (UNG) and endonuclease IV (endo TV), which are available from EPICENTRE Technologies (Madison, WI, USA).
  • UNG uracil-N-glycosylase
  • endonuclease IV endo TV
  • UNG hydrolyzes the N- glycosidic bond between the deoxyribose sugar and uracil in single- and double-sfranded DNA that contains uracil in place of thymidine. UNG has no activity on dUTP or in cleaving uracil from UMP residues in RNA. Endo IV cleaves the phosphodiester linkage at the abasic site. It may be useful to use a thermolabile UNG (e.g., HKTM-UNG from EPICENTRE Technologies, Madison, Wisconsin, USA) for some applications.
  • a thermolabile UNG e.g., HKTM-UNG from EPICENTRE Technologies, Madison, Wisconsin, USA
  • the 3 '-end of a first-strand cDNA that is to become the template sequence for a transcription reaction can be defined by first amplifying the target nucleic acid sequence using any suitable amplification method, such as but not limited to PCR or RT-PCR, that delimits the end sequence.
  • a 3 ' -end of a target sequence need not be at an exact location, and can be random or imprecise, which is the case in some embodiments ofthe invention, there are a number of other methods that can be used for making smaller fragments of a DNA molecule, whether for a target nucleic acid, a target sequence, or otherwise.
  • a target nucleic acid can be fragmented by physical means, such as by movement in and out of a syringe needle or other orifice or by sonication.
  • the ends of physically fragmented double-sfranded DNA can be made blunt prior to denaturation and use in an assay or method ofthe present invention using a T4 DNA polymerase or a kit, such as the End-ItTM DNA End Repair Kit (EPICENTRE Technologies, Madison, WI, USA).
  • a target nucleic acid comprising a target sequence is short enough so that its 3'-end will easily be released from the catenated circular molecules that result from ligation of a bipartite target probe annealed to the target sequence
  • the present invention also includes embodiments of methods, assays, compositions and kits for detecting target sequences comprising larger target nucleic acids, wherein the catenated ligation product is not substantially released from the target nucleic acid.
  • the invention comprises additional steps for release ofthe catenated circular ligation product after annealing and ligation on the target sequence.
  • Baner, J. et al. (Nucleic Acids Research, 26: 5073-5078, 1998, inco ⁇ orated herein by reference) showed that ligation of a linear DNA having two target-complementary end sequences that anneal adjacently on a target sequence resulted in a catenated molecule that was not efficiently replicated by rolling circle replication using phi29 DNA polymerase unless there was a free 3'-end ofthe target nucleic acid near the ligation site.
  • Baner et al. showed that, in order to obtain efficient rolling circle replication by phi29 DNA polymerase of circular ssDNA molecules that had been ligated on a target sequence, the topological link ofthe circular DNA with the target molecules needed to be released.
  • Patent No. 6,558,928, inco ⁇ orated herein by reference provided methods for release of catenated circular DNA molecules in order to improve the efficiency of rolling circle replication reactions.
  • the present invention comprises the use ofthe methods described in U.S. Patent No. 6,558,928, which methods are inco ⁇ orated herein by reference, in order to release catenated circular ligation products for rolling circle franscription as described herein.
  • the circular ssDNA ligation product is released from catenation with the target sequence by digestion with an exonuclease after ligation ofthe bipartite probe on the target sequence.
  • Preferred exonucleases are those that digest single- stranded DNA and that do not have endonuclease activity.
  • One enzyme that can be used is exonuclease I (exo I) (EPICENTRE Technologies, Madison, WI), which has 3'-to-5' single- sfranded exonuclease activity in the presence of Mg 2+ cations.
  • exonuclease VII Another enzyme that can be used is exonuclease VII (exo VII) (EPICENTRE Technologies, Madison, WI), which has both 3'-to-5' and 5'-to-3' single-sfranded exonuclease activity.
  • Exo VII is active in the absence of Mg 2+ cations, which makes it a preferred embodiment for many applications.
  • Rec J nuclease (EPICENTRE Technologies, Madison, WI), which has 5'-to-3' single-sfranded exonuclease activity in the presence of Mg 2+ cations, can also be used in some embodiments.
  • a double-stranded exonuclease such as but not limited to exonuclease Til (exo III) in addition to a single-stranded exonuclease, such as but not limited to exo I, in order to release catenated circular DNA molecules from a target nucleic acid comprising a target sequence.
  • the target sequence that has been digested with an exonuclease can prime rolling circle replication after exonuclease removal ofthe non-base-paired 3' end.
  • DNA polymerases are used in some embodiments ofthe present invention in order to fill by DNA polymerase extension one or more "gaps" between non-contiguous target- complementary sequences of target probes that are annealed to a target sequence.
  • the invention is not limited to a particular DNA polymerase to accomplish this pu ⁇ ose, and the invention includes use of any DNA polymerase that is active in filling a gap under suitable reaction conditions.
  • a suitable DNA polymerase fills the gap by DNA polymerase extension from the 3'- hydroxyl end of one target-complementary target probe to the 5 '-end ofthe next target- complementary target probe, without strand-displacement ofthe target-complementary 5'-end portion of a target probe.
  • the "strand displacement" activity of a DNA polymerase is an operational definition and depends on reaction conditions, such as, but not limited to, reaction temperature, buffer, salt concentration, pH, Mg 2+ concentration, use of cosolvents such as DMSO, or DNA polymerase enhancers such as betaine, as well as on the intrinsic properties of a DNA polymerase.
  • reaction conditions such as, but not limited to, reaction temperature, buffer, salt concentration, pH, Mg 2+ concentration, use of cosolvents such as DMSO, or DNA polymerase enhancers such as betaine, as well as on the intrinsic properties of a DNA polymerase.
  • reaction conditions such as, but not limited to, reaction temperature, buffer, salt concentration, pH, Mg 2+ concentration, use of cosolvents such as DMSO, or DNA polymerase enhancers such as betaine, as well as on the intrinsic properties of a DNA polymerase.
  • cosolvents such as DMSO
  • DNA polymerase enhancers such as betaine
  • Sfrand displacement and DNA polymerase processivity can be assayed using methods described in Kong et al. (J. Biol. Chem., 268: 1965-1975, 1993) and references cited therein, all of which are inco ⁇ orated herein by reference.
  • Preferred DNA polymerases lack 5'-to'3' and 3'-to-5' exonuclease activity under the reaction conditions used.
  • the DNA polymerase used to fill a gap lacks a 5' structure-dependent nuclease, such as CleavaseTM or InvaderTM nucleases used by Third Wave Technologies (Madison, WI) because these enzymes could cleave off an unpaired nucleotide, especially at the 5'-end of a sequence that is partially annealed to a target sequence 3'-of another target probe, and then the DNA polymerase could fill in the gap formed. Therefore, a DNA polymerase with 5' structure-dependent nuclease activity could result in inaccurate results in the assay.
  • a 5' structure-dependent nuclease such as CleavaseTM or InvaderTM nucleases used by Third Wave Technologies (Madison, WI
  • DNA polymerases are thermostable so that activity is more consistent during the course of a method or assay ofthe invention and in order to be more easily stored without loss of polymerase activity.
  • a preferred DNA polymerase ofthe invention for filling gaps between target probes annealed to a target sequence is T4 DNA polymerase.
  • Another DNA polymerase that can be used is T7 DNA polymerase (EPICENTRE Technologies, Madison, WI, USA).
  • RNA polymerase RNA polymerase
  • the invention comprises the use of any RNA polymerase that Can be used to make a franscription product using a transcription substrate obtained using the methods ofthe invention described herein.
  • a cognate transcription promoter In order to make a franscription substrate ofthe invention, a cognate transcription promoter must be known and obtained, wherein the RNA polymerase recognizes and binds to said promoter with specificity and initiates franscription therefrom.
  • a "cognate promoter" for a particular RNA polymerase is a promoter that is recognized by that particular RNA polymerase with specifity.
  • RNA polymerase for a particular promoter is one that recognizes the particular promoter with substantially greater specifity than another RNA polymerase that recognizes one or more other promoter sequences, thus permitting transcription ofthe template that is joined with the promoter with specificity even in the presence of other sequences that are not recognized as a promoter by the particular RNA polymerase.
  • a "transcription promoter” or a “promoter sequence” or a “promoter” is a specific nucleic acid sequence that is recognized by a DNA-dependent RNA polymerase (or simply an "RNA polymerase” or "RNAP" ofthe invention as a signal to bind to the nucleic acid and begin the transcription of RNA at a specific site.
  • RNA polymerases that recognize a double-stranded sequence as the promoter sequence.
  • T7-type RNA polymerases E. coli RNAP, Thermus thermophilus RNAP, mitochondrial RNA polymerases and eukaryotic RNA polymerases
  • RNA polymerases that recognize a double-sfranded promoter it will be understood that, when one refers to "a promoter sequence,” the promoter comprises both that sequence and its complementary sequence.
  • the promoter sequence that is joined to the template sfrand for transcription is referred to as the "sense promoter sequence” and its complementary sequence is referred to as the "anti-sense promoter sequence.”
  • a functional double-stranded promoter therefore comprises a complex between a sense promoter sequence and an anti-sense promoter sequence, and a franscription substrate for an RNA polymerase that uses a double-sfranded promoter is obtained only after annealing of an anti-sense promoter sequence to a sense promoter sequence that is joined to the 3'-end of a template for franscription.
  • Phage N4 vRNAP and N4 mini-vRNAP deletion mutants are exceptional in that they bind and initiate transcription from single-sfranded promoters.
  • a "promoter sequence" for an N4 vRNAP or mini-vRNAP comprises only a single-sfranded sense promoter sequence that is joined to the 3'-end of a template for transcription, which comprises a functional "franscription substrate.” Still further, the invention also comprises embodiments that use single-sfranded “pseudopromoters” or “synthetic promoters.” As discussed in greater detail elsewhere herein, pseudopromoters are single-sfranded sense promoter sequences that are artificially obtained by an in vitro process of "molecular evolution” and selection of a sequence for promoter activity for a particular RNA polymerase.
  • a pseudopromoter is a sequence that is able to serve the same function as a single-sfranded sense promoter like an N4 promoter for its cognate RNAP.
  • R polymerases and their cognate promoters are known in the art. For example, Inspection ofthe sequences of phage, archaebacterial, eubacterial, eukaryotic and viral DNA-dependent RNA polymerases has revealed the existence of two enzyme families.
  • RNA polymerases are complex multisubunit enzymes (5-14 subunits) composed of two large subunits, one to several subunits of intermediate molecular weight (30-50-kDa) and none to several subunits of small molecular weight ( ⁇ 30-kDa) (Archambault and Friesen, Microbiol. Rev. 57:703-724, 1993; Record et al., Cell and Molecular Biology 1:792-821, 1995.
  • Eubacterial RNA polymerases are the simplest with an ⁇ / 3/3' core structure.
  • RNA polymerases consist of a single (-100 kDa) polypeptide which catalyzes all functions required for accurate franscription (Cheetham, et al., Curr. Op. In Sfruc. Biol. 10: 117-123, 2000).
  • heterodimeric bacteriophage N4 RNAP II, nuclear-coded mitochondrial, and Arabidopsis chloroplast RNA polymerases show sequence similarity to the phage RNA polymerases (Cermakian, et al., Nuc. Acids Res. 24:648-654, 1996; Hedtke, et al., Science 277:809-811, 1997; Zehring, et al, J. Biol. Chem. 258:8074-8080, 1983).
  • T7 RNA polymerase contains additional structures dedicated to nascent RNA binding, promoter recognition, dsDNA unwinding and RNA:DNA hybrid unwinding (Cheetham, et al., Curr. Op. In Sfruc. Biol. 10:117-123, 2000; Sousa, Trends in Biochem. Sci. 21:186-190, 1996).
  • This unwinding activity of T7 RNAP and T7-like RNAPs is described in Japanese Patent Nos. JP4304900 and JP4262799 as "helicase-like activity.”
  • RNA polymerases recognize specific sequences, called promoters, on B form double-stranded DNA.
  • Eubacterial promoters (except those recognized by ⁇ 54 ) are characterized by two regions of sequence homology: the -10 and the -35 hexamers (Gross, et al., Cold Spring Harbor Symp. Quant. Biol. 63:141-156, 1998). Specificity of promoter recognition is conferred to the core enzyme by the ⁇ subunit, which makes specific interactions with the -10 and -35 sequences through two distinct DNA binding domains (Gross, et al., Cold Spring Harbor Symp. Quant. Biol. 63:141-156, 1998).
  • This modular promoter structure is also present at the promoters for eukaryotic RNA polymerases I, II and III. Transcription factors TFIIIA and TFIIIC direct recognition of RNAP III to two separate sequences (boxes A and C, separated by defined spacing) at the 5S gene promoter, while transcription factors TFIHB and TFIIIC direct recognition of this enzyme to blocks A and B, separated by variable distance (31-74 bp) at the tRNA promoters (Paule, et al., Nuc. Acids Res. 28:1283-1298, 2000).
  • RNAP I franscription initiation at the human rRNA promoters are also restricted to two regions: the "core" region located at -40 to +1 and the "upstream” region present at -160 to -107 (Paule, et al., Nuc. Acids Res. 28:1283-1298, 2000). Assembly ofthe initiation complex at RNAP H promoters requires several general transcription factors (TFIIA, TFIB, TFIID, TFIIE, TFIIF and TFIIH). Recognition involves three core elements: the TATA box located at position -30 and recognized by TBP, the initiator element located near -1, and the downstream promoter element near +30 (Roeder, Trends Biochem. Sci. 21:327-335, 1996).
  • RNAP promoters span a continuous highly conserved 23 bp region extending from position -17 to +6 relative to the start site of transcription (+1) (Rong, et al., Proc. Natl. Acad. Sci. USA 95:515-519, 1998).
  • the yeast mitochondrial RNAP promoters are even smaller, extending from -8 to +1 (Shadel, et al., J. Biol. Chem. 268:16083-16086, 1993).
  • One exception are the promoters for N4 RNAP II, which are restricted to two blocks of conserved sequence: a/tTTTA at +1 and AAGACCTG present 18-26 bp upstream of +1 (Abravaya, et al., J. Mol. Biol. 211:359-372, 1990).
  • RNA polymerases The activity of the multisubunit class of RNA polymerases is enhanced by activators at weak promoters. Transcription activators generally bind at specific sites on double- sfranded DNA upsfream ofthe -35 region (with the exception ofthe T4 sliding clamp activator), or at large distances in the cases of enhancers (Sanders, et al., EMBO Journal 16:3124-3132, 1997). Activators modulate franscription by increasing the binding (formation of closed complex) or isomerization (formation of open complex) steps of transcription through interactions with the or ⁇ subunits of RNAP (Hochschild, et al., Cell 92:597-600, 1998).
  • N4SSB the activator of E. coli RNAP ⁇ 70 at the bacteriophage N4 late promoters, which activates transcription through direct interactions with the ⁇ ' subunit of RNAP in the absence of DNA binding
  • Proteins that bind to ssDNAs with high affinity but without sequence specificity have been purified and characterized from several prokaryotes, eukaryotes, and their viruses (Chase, et al, Ann. Rev. Biochem. 55:130-136, 1986).
  • SSBs proteins
  • Binding to DNA results in the removal of hai ⁇ in structures found on ssDNA, providing an extended conformation for proteins involved in DNA metabolism.
  • Several lines of evidence suggest that single-sfranded DNA binding proteins play a more dynamic role in cellular processes. Genetic and biochemical evidence indicates that these proteins are involved in a multitude of protein-protein interactions including franscription activation (Rothman-Denes, et al., Genes Devepmnt. 12:2782-2790, 1999).
  • N4 virion RNA polymerase Bacteriophage N4 virion RNA polymerase (N4 vRNAP) is present in N4 virions and is injected into the E. coli cell at the beginning of infection, where it is responsible for franscription of theN4 early genes (Falco, et al., Proc. Natl. Acad. Sci. (USA) 74:520-523, 1977; Falco, et al., Virology 95:454-465, 1979; Malone, et al., Virology 162:328-336, 1988).
  • the N4 vRNAP gene maps to the late region ofthe N4 genome (Zivin, et al., J. Mol. Biol. 152:335-356, 1981).
  • N4 vRNAP purified from virions is composed of a single polypeptide with an apparent molecular mass of approximately 320,000 kDa (Falco, et al., Biol. Chem.255:4339- 4347, 1980).
  • N4 vRNAP recognizes promoters on single-sfranded templates (Falco, et al., Proc. Natl. Acad. Sci. USA 75:3220-3224, 1978). These promoters are characterized by conserved sequences and a 5 bp stem, 3 base loop hai ⁇ in structure (FIG.
  • N4 vRNAP lacks unwinding or helicase-like activity on dsDNA and also lacks unwinding activity on RNA:DNA hybrids. In vivo, E. coli gyrase and single-sfranded binding protein are required for franscription by N4 vRNAP (Falco, et al., J. Biol. Chem. 255:4339- 4347, 1980; Markiewicz, et al., Genes andDev. 6:2010-2019, 1992).
  • RNA synthesis requires RNA polymerase, a DNA template, an activated precursor (the ribonucleoside triphosphates ATP, GTP, UTP and CTP (XTP)), and divalent metal ions such as Mg +2 or Mn +2 .
  • the metal ion Mg +2 is sfrongly preferred.
  • Synthesis of RNA begins at the promoter site on the DNA. This site contains a sequence which the RNA polymerase recognizes and binds. The RNA synthesis proceeds until a termination site is reached.
  • N4 vRNAP termination signals comprise a hai ⁇ in loop that forms in the newly synthesized RNA which is followed by a string of uracils (poly U).
  • N4 vRNAP termination signals possess all ofthe characteristics of eubacterial sequence- dependent terminators.
  • Single-stranded DNA of varying lengths can be used as a template for RNA synthesis using the N4 vRNAP or mini-vRNAP.
  • EcoSSB is essential for N4 vRNAP franscription in vivo (Falco et al., Proc. Natl. Acad. Sci. (USA) 75:3220-3224, 1978; Glucksmann, et al., Cell 70:491-500, 1992).
  • EcoSSB is a specific activator of N4 vRNAP on single-sfranded and supercoiled double-stranded DNA templates.
  • RNA polymerases of the invention are T7 RNAP (e.g., see Studier, FW et al., pp. 60-89 in Methods in Enzymology, Vol.
  • T7-like bacteriophage ed. by Goeddel, DV, Academic Press, 1990, inco ⁇ orated herein by reference
  • T7-type RNA polymerases other "T7-like" or "T7-type” RNA polymerases.
  • the genetic organization of all T7-like bacteriophage that have been examined has been found to be essentially the same as that of T7.
  • T7-like bacteriophages include, but are not limited to Escherichia coli phages T3, phi I, phi II, W31, H, Y, Al, 122, cro, C21, C22, and C23; Pseudomona putida phage gh-1; Salmonella typhimurium phage SP6; Serratia marcescens phages IV; Citrobacter phage Villl; and Klebsiella phage No. 11 (Hausmann, Current Topics in Microbiology and Immunology 75:77-109, 1976; Korsten et al., J. Gen. Virol.
  • T7 RNAP Y639F mutant enzyme such as, but not limited to, T7 RNAP Y639F mutant enzyme, T3 RNAP Y573F mutant enzyme, SP6 RNAP Y631F mutant enzyme, T7 RNAP having altered amino acids at both positions 639 and 784, T3 RNAP having altered amino acids at both positions 573 and 785, or SP6 RNAP having altered amino acids at both positions 631 and 779 can also be used in some embodiments of methods or assays ofthe invention.
  • modified RNA molecules that contain 2'-F-dCMP and 2'-F- dUTP are resistant to RNase A-type ribonucleases (Sousa et al., U.S. Patent No. 5,849,546, inco ⁇ orated herein by reference); (ii) can be delivered into cells without complexing with a fransfection agent and in the presence of serum (Capodici et al., J.
  • RNA polymerase may be present in a reaction in addition to an RNA polymerase ofthe invention.
  • a mutant RNAP enzyme is not preferred.
  • the dNTPs in a reaction mixture as subsfrates for a DNA polymerase can be inco ⁇ orated into a transcription product by the mutant RNAP enzyme, although less efficiently than an NTP.
  • Promoter sequences may be used that that are recognized specifically by a DNA-dependent RNA polymerase, such as, but not limited to, those described by Chamberlin and Ryan, In: The Enzymes. San Diego, Calif., Academic Press, 15:87-108, 1982, and by Jorgensen et al., J. Biol. Chem. 266:645-655, 1991.
  • RNA polymerase promoter sequences are especially useful, including, but not limited to, promoters derived from SP6 (e.g., Zhou and Doetsch, Proc. Nat. Acad. Sci.
  • T7 e.g., Martin, and Coleman, Biochemistry 26:2690-2696, 1987
  • T3 e.g., McGraw et al., Nucl. Acid. Res. 13:6753-6766, 1985.
  • the length ofthe promoter sequence will vary depending upon the promoter chosen.
  • the T7 RNA polymerase promoter can be only about 25 bases in length and act as a functional promoter, while other promoter sequences require 50 or more bases to provide a functional promoter.
  • RNA polymerase promoter sequence that can be used is derived from
  • Thermus thermoph ⁇ lus see, e.g., Wendt et al, Eur. J. Biochem. 191:467-472, 1990; Faraldo et al., J. Bact. 174:7458-7462, 1992; Hartmann et al., Biochem. 69:1097-1104, 1987; Hartmann et al., Nucl. Acids Res. 19:5957-5964, 1991) with the cooesponding Thermus thermophilus RNAP (EPICENTRE Technologies, Madison, WI).
  • the promoters ofthe invention comprise single- sfranded pseudopromoters or synthetic promoters that are recognized by an RNAP so as to function in a method or assay ofthe invention.
  • a "pseudopromoter” or “synthetic promoter” of the present invention can be any single-sfranded sequence that is identified and/or selected to be functional as a promoter for in vitro transcription by an RNAP that recognizes said promoter with specificity and which functions as a promoter for said RNAP in a method or assay ofthe invention.
  • a promoter comprising a pseudopromoter or synthetic promoter ofthe invention can be made as described by Ohmichi et al. (Proc. Natl. Acad. Sci. USA. 99: 54-59, 2002), which reference is inco ⁇ orated herein by reference. If a pseudopromoter or synthetic promoter is used as a promoter in a method or assay ofthe invention, then the corresponding RNAP for which the pseudopromoter or synthetic promoter was identified and or selected is used in the method or assay.
  • a target probe with a promoter comprising a ssDNA pseudopromoter can be obtained and used in a method or assay ofthe invention that uses E.
  • coli RNAP or a T7-type phage RNAP such as, but not limited to, T7 RNAP, T3 RNAP, or SP6 RNAP, as described by Ohmichi et al. (Proc. Natl. Acad. Sci. USA. 99: 54-59, 2002) and inco ⁇ orated herein by reference.
  • Suitable pseudopromoters for E. coli RNAP that can be used in embodiments ofthe present invention are those found by Ohmichi et al. (Proc. Natl. Acad. Sci. USA. 99: 54-59, 2002).
  • a single-sfranded promoter can also comprise a single-sfranded N4 vRNAP promoter (Haynes, et al., Cell 41:597-605, 1985; Glucksmann, et al., Cell 70:491-500, 1992), such as aPl promoter (3'-CAACGAAGCGTTGAATACC T-5'), aP2 promoter (3'-TTCTTCGAGGCGAAGAAAACCT -5') or aP3 promoter (3'-CGACGAGGCGTCGAAAACCA-5') in some embodiments, in which case a transcriptionally active 1,106-amino acid domain ofthe N4 vRNAP ("mini-vRNAP"), which corresponds to amino acids 998-2103 of N4 vRNAP (Kazmierczak, K.M., et al., EMBO J., 21: 5815-5823, 2002; U.S.
  • aPl promoter 3'-CAACGAAGCGTTGA
  • Patent Application No. 20030096349 inco ⁇ orated herein by reference
  • an N4 mini-vRNAP Y678F mutant enzyme U.S. Patent Application No. 20030096349
  • a single-stranded promoter is used, the cognate RNA polymerase for the promoter is used for transcription, but an anti-sense promoter oligo is not needed to obtain a circular or linear franscription subsfrate in those embodiments.
  • Use of compositions comprising a single-sfranded promoter is not suitable for embodiments ofthe invention in which an anti-sense promoter oligo that is attached to a solid support is used to obtain a transcription substrate comprising a double-sfranded promoter.
  • the promoter sequence that is joined to the 3'-end of the target-complementary sequence at the 5'-end of a monopartite promoter target probe or a bipartite target probe ofthe present invention, as described in greater detail elsewhere herein, comprises a "sense promoter sequence” or a "sense promoter.” If the RNA polymerase used in a method ofthe invention requires a double-sfranded promoter to obtain a functional promoter, the "sense promoter sequence” refers to the promoter sequence ofthe double-sfranded promoter that is operably joined to the template strand for transcription (i.e., the sfrand that is copied to make a franscription product).
  • the sense promoter sequence that is joined to a target- complementary sequence in a target probe can also include one, two or a small number of additional nucleotides that serve as sites for initiation of franscription, which nucleotides are designated respectively as "the +1 nucleotide,” "the +2 nucleotide,” etc.
  • a co ⁇ esponding sense T7 promoter sequence and +1 base that can be used in a target probe ofthe present invention is: [00197] (5' CTATAGTGAGTCGTATTA 3').
  • a corresponding anti-sense promoter oligo can comprise the following anti-sense T7 promoter sequence and +1 base to obtain a functional double-sfranded promoter sequence for T7 RNAP:
  • composition ofthe invention can be an anti-sense promoter oligo that is annealed to a complementary sense promoter in order to obtain a circular or linear franscription substrate having a functional double-stranded promoter.
  • an anti-sense promoter oligo comprises deoxyribonucleotides. Modified nucleotides or modified linkages should be used in an anti-sense promoter oligo only after carefully determining that they do not substantially affect the ability ofthe anti-sense promoter oligo to complex with a sense promoter sequence or to bind the RNA polymerase or to affect the ability ofthe RNA polymerase to initiate transcription using the template sfrand.
  • modified nucleotides can be used for a particular pu ⁇ ose.
  • modified linkages such as, but not limited to alpha-thiophosphate sugar linkages that are resistant to certain nucleases can be used for a particular pu ⁇ ose.
  • An anti-sense promoter oligo can be of any length so long as it has sufficient length to comprise an anti-sense promoter sequence that, when annealed to a sense promoter, makes a functional double-stranded promoter that can be used by an RNA polymerase under transcription conditions to make a transcription product.
  • the oligo comprising the anti-sense promoter can comprise additional nucleotides that are 3'-of or 5'-of the anti-sense promoter sequence so long as the additional nucleotides do not bind the intended target sequence or another component used in a method ofthe invention in a manner that is independent of complexing ofthe anti-sense promoter sequence with the sense promoter sequence in a ligation product of target probes annealed to the target sequence, or otherwise negatively affect the results ofthe method.
  • modified nucleotides are used in anti-sense promoter oligo for a pmpose such as, but not limited to for attaching a biotin or other moiety, it is preferred that the modified nucleotide is in a nucleotide that does not comprise the anti-sense promoter sequence if possible. If an anti-sense promoter oligo is present in a reaction when steps such as primer extension with a DNA polymerase or ligation with a ligase are performed and it is not intended to primer extend the anti-sense promoter oligo annealed to the ligation product, the anti-sense promoter oligo is designed so that it cannot participate in these reactions.
  • an anti-sense promoter oligo that has a dideoxynucleotide or another termination nucleotide on its 3 '-end so that it can't be primer-extended.
  • An anti-sense promoter oligo also typically does not have a phosphate group on its 5'-end so that it cannot participate in a ligation reaction.
  • composition of an anti-sense promoter oligo ofthe invention can be an oligonucleotide comprising an anti-sense promoter that is immobilized or attached to a solid support.
  • a composition ofthe invention can be an anti- sense promoter oligo having a moiety, such as but not limited to a biotin moiety that permits binding ofthe anti-sense promoter oligo to a solid support after annealing to the ligation product obtained by ligation of target probes annealed to a target sequence. Annealing ofthe resulting ligation product to the anti-sense promoter oligo thus generates a franscription substrate ofthe present invention.
  • An anti-sense promoter oligo that is to be attached to a solid support can comprise a biotin moiety at or near its 5'-end, in which case, the anti-sense promoter oligo can be attached to a solid support that is covalently or non- covalently joined to an avidin or streptavidin moiety using any ofthe variety of joining methods known in the art.
  • the anti-sense promoter oligo is attached to a solid support prior to annealing to the ligation product or is attached to the solid support after annealing to the ligation product
  • the anti-sense promoter oligo comprising the anti-sense promoter is immobilized on the solid support at or near its 5'-end and the anti-sense promoter sequence is at a sufficient distance from the surface ofthe solid support so that the sense promoter in a circular ligation product resulting from ligation of a bipartite target probe annealed to a target sequence or in a linear ligation product resulting from ligation of monopartite target probes annealed to a target sequence can complex with or anneal to the anti-sense sequence so as to make a functional immobilized circular or linear transcription substrate, respectively, when the support is incubated with an RNA polymerase that uses the double-sfranded promoter to make a franscription product in a reaction medium under suitable franscription conditions
  • a biotin may be attached to the anti-sense promoter oligo, for example, but without limitation, by using a ribonucleoside triphosphate that is derivatized with biotin.
  • a ribonucleoside triphosphate that is derivatized with biotin.
  • Exemplary methods for making derivatized nucleoside triphosphates are disclosed in detail in Rashtchian et al., "Nonradioactive Labeling and Detection of Biomolecules," C. Kessler, Ed., Springer- Verlag, New York, pp. 70- 84, 1992, herein inco ⁇ orated by reference.
  • the solid support has a chemical composition and structure so that it does not non-specifically bind nucleic acid from a sample or that comprises a composition ofthe invention, such as, but not limited to a sense promoter primer.
  • the solid support has a chemical composition and structure so that it does not non-specifically bind enzymes, co-factors or other substances in reactions comprising methods ofthe invention.
  • solid supports can comprise dipsticks, membranes, such as nitrocellulose or nylon membranes, beads, chips or slides used for making arrays or microarrays, and the like.
  • solid supports which can be used for the present invention are also known in the art and can be used. Numerous other methods for attaching a molecule comprising an oligonucleotide to a surface or other substance are known in the art, and any known method for attaching or immobilizing a molecule comprising an anti-sense promoter oligo can be used to make a composition comprising an immobilized anti-sense promoter oligo is included in the present invention.
  • a single-sfranded promoter such as the P2 promoter for an N4 mini-vRNAP
  • the reaction conditions for in vitro transcription are those provided with the AmpliScribeTM T7-FlashTM Transcription Kit, or the AmpliScribeTM T7 High Yield Transcription Kit, or the DuraScribeTM T7 Transcription Kit or, for inco ⁇ oration of 2'-substituted deoxyribonucleotides other than 2'- fluorine-substituted deoxyribonucleotides, with the T7 R&DNATM Polymerase, in each case according to the instructions ofthe manufacturer EPICENTRE Technologies, Madison, WI).
  • the reaction conditions for in vitro transcription are those provided with the AmpliScribeTM T3 High Yield Transcription Kit or with the AmpliScribeTM T3-FlashTM High Yield Transcription Kit, or with the AmpliScribeTM SP6 High Yield Transcription Kit, in each case according to the instructions ofthe manufacturer EPICENTRE Technologies, Madison, WI). Kits or individual enzymes, including reaction buffers and instructions for use are also available. For example, products are commercially available for E. coli RNA polymerase and Thermus RNAP (EPICENTRE Technologies, Madison, WI).
  • RNA polymerase a particular RNA polymerase.
  • the conditions below can be used for in vitro franscription with T7 RNAP, an exemplary T7-like RNAP.
  • An in vitro franscription reaction is prepared by setting up a reaction mixture containing the following final concentrations of components, added in the order given: 0.1 micromolar of a T7 RNAP sense promoter-containing DNA oligo; IX transcription buffer comprising 40 mM Tris-HCl (pH 7.5), 6 M MgCl 2 , 2 mM spermidine, and 10 mM NaCl; 1 mM DTT; 0.5 mM of each NTP (ATP, CTP, GTP and UTP); deionized RNase-free water so the final volume will be 50 microliters after addition of an RNAP.
  • 2'-F-dUTP and 2'-F-dCTP are used at a final concentration of 0.5 mM each in place of UTP and CTP in order to obtain synthesis of modified RNA which is resistant to ribonuclease A-type enzymes.
  • An RNase inhibitor such as placental RNase inhibitor or an antibody inhibitor, which are commercially available, can be added to the reaction.
  • Inorganic pyrophosphatase can be added to the reaction to prevent pyrophosphorolysis ofthe franscription product.
  • Other modified nucleoside triphosphates can be used in place of or in addition to the canonical NTPs for specific applications.
  • the reaction mixture is then incubated at 37° C to permit synthesis of RNA from the template.
  • the reaction can be followed by gel electrophoresis on a PAGE gel.
  • the invention is not limited to these reaction conditions or concentrations of reactants. Transcription reaction conditions can be altered to accommodate reactions conditions for other enzymes and reactants used in a method. Preferred conditions of a transcription process herein include a pH of between 6 and 9, with a pH of between 7.5 and 8.5 more preferred. Mg +2 or Mn +2 , preferably Mg +2 may be admixed. Preferred temperatures for the reaction are 25° C to 50° C with the range of 30° C to 45° C being more preferred and the range of 32° C to 42° C being most preferred. Those with skill in the art will know that other suitable reaction conditions under which an RNA polymerase ofthe invention can be used can be found by simple experimentation, and any of these reaction conditions are also included within the scope ofthe invention.
  • mini-vRNAP or mini-vRNAP Y678F enzymes are used for in vitro franscription of a transcription subsfrate having a single-stranded N4 promoter
  • the following in vitro franscription reaction can be prepared by setting up a reaction mixture containing the following final concenfrations of components, added in the order given: 0.1 micromolar of a N4 vRNAP promoter-containing DNA oligo; 1.0 micromolar EcoSSB Protein; IX transcription buffer comprising 40 mM Tris-HCl (pH 7.5), 6 mM MgCl 2 ., 2 mM spermidine, and 10 mM NaCl; 1 mM DTT; 0.5 mM of each NTP (ATP, CTP, GTP and UTP); deionized RNase-free water so the final volume will be 50 microliters after addition of an RNAP; and 0.1 micromolar of mini-vRNAP or mini-vRNAP Y678F enzyme
  • 2'-F- dUTP and 2'-F-dCTP are used at a final concentration of 0.5 mM each.in place of UTP and CTP in order to obtain synthesis of modified RNA which is resistant to ribonuclease A-type enzymes.
  • Other modified nucleoside triphosphates can also be used in place of or in addition to the canonical NTPs for specific applications.
  • the reaction mixture is then incubated at 37° C to permit synthesis of RNA from the template.
  • the reaction can be followed by gel electrophoresis on a PAGE gel.
  • Other components and reaction conditions, including those discussed above for T7-type RNA polymerases, can also be used without undue experimentation those with knowledge in the art to obtain suitably optimized conditions.
  • RNA can comprise RNA or, in view of the ability of certain polymerases ofthe invention, including, without limitation, a T7 RNAP Y639F mutant enzyme or a T7 RNAP mutant enzyme having altered amino acids at both positions 639 and 784 (Sousa et al., U.S. Patent No.
  • a transcription product can comprise, in addition to RNA, DNA or modified DNA, or modified RNA, or a mixture thereof.
  • the synthesized transcription product may comprise a detectable label such as a fluorescent tag, biotin, digoxigenin, 2'-fluoro nucleoside triphosphate, or a radiolabel such as a 35 S- or 32 P-label.
  • the synthesized franscription product may be adapted for use as a probe for blotting experiments or in-situ hybridization.
  • Nucleoside triphosphates (NTPs) or derivatized NTPs may be inco ⁇ orated into the transcription product, and may optionally have a detectable label.
  • 5 '-end of a promoter target probe is a template for transcription by the cognate RNA polymerase that recognizes the promoter.
  • the target-complementary sequence at the 5 '-end of a bipartite target probe or a promoter target probe is kept short, with only about 4 to about 100 nucleotides and preferably with about 8 to about 30 nucleotides and the reaction conditions of the method are adjusted to minimize the amount of transcription product obtained in the absence of a target sequence.
  • no transcription product is synthesized using the target- complementary sequence that anneals to the 3 '-end ofthe target sequence or the signal sequence of a target probe as a template since these sequences are not joined to the promoter in the absence of a target sequence.
  • a method ofthe present invention detects, directly or indirectly, synthesis of franscription product that is complementary to the target-complementary sequence at the 3 '-end of a bipartite target probe or, when monopartite target probes are used, that is joined to the 5 '-end of a promoter target probe.
  • Yet another aspect ofthe current invention comprises delivering a transcription product into a cell after transcription.
  • the delivery may be by microinjection, transfection, electroporation or another method in the art.
  • the franscription product comprises RNAi.
  • the franscription products made as a result of an assay or method ofthe invention are detected without separating the transcription products from other reaction components.
  • a variety of separation and detection methods can be used.
  • amplification products are separated by agarose, agarose- acrvlamide or nolvacrvlamide eel electroohoresis usine standard methods.
  • chromatography There are many kinds of chromatography which may be used in the present invention: adso ⁇ tion, partition, ion-exchange and molecular sieve, and many specialized techniques for using them including column, paper, thin-layer and gas chromatography.
  • molecules such as but not limited to franscription subsfrates, transcription products, analyte-binding substances or analytes that are labeled, such as but not limited to biotin-labeled or antigen-labeled, can be captured with beads bearing avidin or antibody, respectively.
  • Microfluidic techniques include separation on a platform such as microcapillaries, designed by ACLARA BioSciences Inc., or the LabChipTM "liquid integrated circuits" made by Caliper Technologies Inc. These microfluidic platforms require only nanoliter volumes of sample, in contrast to the microliter volumes required by other separation technologies. Miniaturizing some ofthe processes involved in genetic analysis has been achieved using microfluidic devices. For example, published PCT Application No. WO 94/05414, to Northrup and White, inco ⁇ orated herein by reference, reports an integrated micro- PCR apparatus for collection and amplification of nucleic acids from a specimen. U.S. Pat. Nos.
  • micro capillary arrays are contemplated to be used for the analysis.
  • Microcapillary array elecfrophoresis generally involves the use of a thin capillary or channel which may or may not be filled with a particular separation medium. Elecfrophoresis of a sample through the capillary provides a size based separation profile for the sample. Microcapillary array elecfrophoresis generally provides a rapid method for size-based sequencing, PCR product analysis and restriction fragment sizing. The high surface to volume ratio of these capillaries allows for the application of higher electric fields across the capillary without substantial thermal variation across the capillary, consequently allowing for more rapid separations.
  • these methods provide sensitivity in the range of attomoles, which is comparable to the sensitivity of radioactive sequencing methods.
  • these methods comprise photolithographic etching of micron scale channels on a silica, silicon or other crystalline subsfrate or chip, and can be readily adapted for use in the present invention.
  • the capillary arrays may be fabricated from the same polymeric materials described for the fabrication ofthe body ofthe device, using the injection molding techniques described herein.
  • Rectangular capillaries are known as an alternative to the cylindrical capillary glass tubes. Some advantages of these systems are their efficient heat dissipation due to the large height-to-width ratio and, hence, their high surface-to-volume ratio and their high detection sensitivity for optical on-column detection modes. These flat separation channels have the ability to perform two-dimensional separations, with one force being applied across the separation channel, and with the sample zones detected by the use of a multi-channel array detector.
  • the capillaries e.g., fused silica capillaries or channels etched, machined or molded into planar substrates, are filled with an appropriate separation/sieving matrix.
  • sieving matrices include, e.g., hydroxyethyl cellulose, polyacrylamide, agarose and the like.
  • the specific gel matrix, running buffers and running conditions are selected to maximize the separation characteristics of the particular application, e.g., the size ofthe nucleic acid fragments, the required resolution, and the presence of native or undenatured nucleic acid molecules.
  • running buffers may include denaturants, chaofropic agents such as urea or the like, to denature nucleic acids in the sample.
  • Mass spectrometry provides a means of "weighing" individual molecules by ionizing the molecules in vacuo and making them “fly” by volatilization. Under the influence of combinations of electric and magnetic fields, the ions follow trajectories depending on their individual mass (m) and charge (z). For low molecular weight molecules, mass spectrometry has been part ofthe routine physical-organic repertoire for analysis and characterization of organic molecules by the determination ofthe mass ofthe parent molecular ion. In addition, by arranging collisions of this parent molecular ion with other particles (e.g., argon atoms), the molecular ion is fragmented forming secondary ions by the so-called collision induced dissociation (CJD).
  • CJD collision induced dissociation
  • “sequencing” had been limited to low molecular weight synthetic oligonucleotides by determining the mass ofthe parent molecular ion and through this, confirming the already known sequence, or alternatively, confirming the known sequence through the generation of secondary ions (fragment ions) via CID in an MS/MS configuration utilizing, in particular, for the ionization and volatilization, the method of fast atomic bombardment (FAB mass spectrometry) or plasma deso ⁇ tion (PD mass spectrometry).
  • FAB mass spectrometry fast atomic bombardment
  • PD mass spectrometry plasma deso ⁇ tion
  • Two ionization deso ⁇ tion techniques are electrospray/ionspray (ES) and matrix- assisted laser deso ⁇ tion/ionization (MALDI).
  • ES electrospray/ionspray
  • MALDI matrix- assisted laser deso ⁇ tion/ionization
  • MALDI mass spectrometry in contrast, can be particularly attractive when a time-of-flight (TOF) configuration is used as a mass analyzer. Since, in most cases, no multiple molecular ion peaks are produced with this technique, the mass spectra, in principle, look simpler compared to ES mass spectrometry. DNA molecules up to a molecular weight of 410,000 Daltons could be desorbed and volatilized.
  • TOF time-of-flight
  • infra red lasers HR infra red lasers HR
  • UV-lasers UV-lasers
  • FET fluorescence energy transfer
  • the excited-state energy ofthe donor fluorophore is transferred by a resonance dipole- induced dipole interaction to the neighboring acceptor. This results in quenching of donor fluorescence.
  • the acceptor is also a fluorophore, the intensity of its fluorescence may be enhanced.
  • the efficiency of energy fransfer is highly dependent on the distance between the donor and acceptor, and equations predicting these relationships have been developed.
  • the distance between donor and acceptor dyes at which energy transfer efficiency is 50% is referred to as the Forster distance (Ro).
  • Other mechanisms of fluorescence quenching are also known including, for example, charge transfer and collisional quenching.
  • WO 96/21144 discloses continuous fluorometric assays in which enzyme-mediated cleavage of nucleic acids results in increased fluorescence. Fluorescence energy fransfer is suggested for use in the methods, but only in the context of a method employing a single fluorescent label which is quenched by hybridization to the target.
  • Signal primers or detector probes which hybridize to the target sequence downstream ofthe hybridization site ofthe amplification primers have been described for use in detection of nucleic acid amplification (U.S. Pat. No. 5,547,861).
  • the signal primer is extended by the polymerase in a manner similar to extension ofthe amplification primers. Extension of the amplification primer displaces the extension product ofthe signal primer in a target amplification-dependent manner, producing a double-stranded secondary amplification product which may be detected as an indication of target amplification.
  • the secondary amplification products generated from signal primers may be detected by means of a variety of labels and reporter groups, restriction sites in the signal primer which are cleaved to produce fragments of a characteristic size, capture groups, and structural features such as triple helices and recognition sites for double-sfranded DNA binding proteins.
  • FITC fluorescein isothiocyanate
  • TRITC teframethylrhodamine isothiocyanate
  • FITC - Texas Red Molecular Probes
  • PYB N-hydroxysuccinimidyl 1-pyrenebutyrate
  • EITC FITC - eosin isothiocyanate
  • PYS N- hydroxysuccinimidyl 1-pyrenesulfonate
  • TAMRA teframethylrhodamine
  • DABYL P-(dimethyl aminophenylazo) benzoic acid
  • EDANS 5-(2'-aminoethyl) aminonaphthalene
  • Any dye pair which produces fluorescence quenching in the detector nucleic acids ofthe invention are suitable for use in the methods ofthe invention, regardless ofthe mechanism by which quenching occurs.
  • Terminal and internal labeling methods are both known in the art and may be routinely used to link the donor and acceptor dyes at their respective sites in the detector nucleic acid.
  • Paul Lizardi discusses the optional use of a transcription promoter in an open circle probe ("OCP") for use in rolling circle amplification ("RCA”), as disclosed in U.S. Patent Nos. 6,344,329; 6,210,884; 6,183,960; 5,854,033; 6,329,150; 6,143,495; 6,316,229; 6,287,824.
  • OCP open circle probe
  • RCA rolling circle amplification
  • a promoter portion can be included in an open circle probe so that RNA transcripts can be generated from tandem sequence DNA (“TS-DNA”), which is a product of rolling circle amplification.
  • the RNA transcripts are primary amplification products and are synthesized by in vitro transcription of transcription subsfrates obtained by target-dependent joining of target probes.
  • the RNA transcripts ofthe present invention are complementary to the target probes used in an assay or method.
  • Preferred promoters in the methods of Lizardi are T7 or SP6 RNA polymerase promoters, which are double-sfranded promoters, and the cognate polymerase for the promoter is used for transcriptional amplification.
  • Lizardi's open circle probes actually contain a protopromoter sequence, to which a complementary sequence must be annealed or a second DNA strand needs to be synthesized in order to obtain a functional promoter.
  • a promoter on an open circle probe if present, is preferably immediately adjacent to the left target probe (i.e., the promoter is 5 '-of the target- complementary sequence on the 3'-end ofthe open circle probe) and is oriented to promote transcription toward the 3'-end ofthe open circle probe so the orientation results in transcripts that are complementary to TS-DNA.
  • a sense promoter sequence of the present invention must be located 3 '-of the target-complementary sequence at the 5 '-end of a bipartite target probe or must be located 3'-of the target-complementary sequence at the 5'-end of a monopartite promoter target probe in order to function in the assays and methods ofthe invention.
  • promoters if present at all, are included in open circle probes for the methods of Lizardi in order to obtain secondary amplification of DNA replication products, rather than for the pvupose of primary amplification as is the case in the methods and assays of the present invention.
  • Toshiya et al. did not disclose target-dependent transcription using monopartite target probes that generate linear transcription substrates in the presence of a target sequence.
  • Japanese Patent Nos. JP4304900 and JP4262799 of Aono Toshiya et al. did not disclose target-dependent transcription using monopartite target probes that generate linear transcription substrates in the presence of a target sequence.
  • Japanese Patent Nos. JP4304900 and JP4262799 of Aono Toshiya et al.
  • Toshiya et al. did not disclose target-dependent transcription using monopartite target probes that generate linear transcription substrates in the presence of a target sequence.
  • JP4304900 and JP4262799 also did not disclose a reaction comprising coupled rolling circle replication and target-dependent franscription, an example of which is shown in Figure 5 herein, wherein a first target sequence amplification probe (TSA probe) is used to amplify the number of target sequences that can serve as annealing and ligation sites for target probes that are used to obtain a franscription substrate for by target-dependent transcription, thus increasing the sensitivity ofthe methods and assays ofthe present invention.
  • TSA probe first target sequence amplification probe
  • Toshiya et al. also did not disclose other methods for amplifying the amount of transcription product obtained, such as the method shown in Figure 9 herein.
  • Toshiya et al. did not disclose the use of an anti-sense promoter oligo that is either attached to a solid support or that has a moiety, such as a biotin moiety, that permits binding to a solid support that is joined to another moiety, such as a sfreptavidin moiety, during the processes of a method or assay ofthe present invention.
  • a moiety such as a biotin moiety
  • the present invention also comprises target-dependent franscription methods that use target probes and target-dependent franscription to detect non-nucleic analytes by detecting a target sequence comprising a target sequence tag that is joined to an analyte-binding substance that binds the analyte.
  • target-dependent franscription methods that use target probes and target-dependent franscription to detect non-nucleic analytes by detecting a target sequence comprising a target sequence tag that is joined to an analyte-binding substance that binds the analyte.
  • JP4304900 and JP4262799 did not disclose methods for detecting analytes other than nucleic acids.
  • the present invention also discloses methods that use a target probe that has a signal sequence such as, but not limited to a sequence for a substrate for Q-beta replicase, that permits a significant additional increase in sensitivity and speed of an assay or method for detecting a target sequence, such as by incubating a transcription product comprising a Q-beta replicase substrate with Q-beta replicase under replication conditions.
  • a signal sequence such as, but not limited to a sequence for a substrate for Q-beta replicase
  • a signal sequence ofthe present invention permits easier detection ofthe franscription product, whether by an indirect means, such as by detecting the amount of a Q-beta subsfrate replicated by Q-beta replicase, or by a direct means, such as using a molecular beacon to detect a specific sequence comprising the transcription product.
  • an indirect means such as by detecting the amount of a Q-beta subsfrate replicated by Q-beta replicase
  • a direct means such as using a molecular beacon to detect a specific sequence comprising the transcription product.
  • Toshiya et al. did not disclose that the target-complementary sequences at the ends ofthe straight chain nucleotide probe of their invention should be adjacent to or joined to the 5'- end of a sense promoter sequence and did not specify the distance ofthe promoter sequence from the target-complementary sequences.
  • the illusfrations of Toshiya et al. show the promoter sequence at some distance from the target-complementary sequences. If the sense promoter sequence is far from the target-complementary sequence, as shown in the figures of Toshiya et al., the amount of background franscription product obtained from the unligated probe will be increased.
  • the present invention discloses that the sense promoter sequence of a bipartite target probe is joined to the 3'-end ofthe target-complementary sequence that anneals to the 5'-end ofthe target sequence, so that background transcription is minimized.
  • the methods ofthe present invention which pertain to the use of bipartite target probes to generate circular transcription substrates also comprise additional embodiments that differ from the methods of Toshiya et al. in certain important ways.
  • the methods disclosed by Toshiya et al. specified that only an RNA polymerase with helicase-like activity should be used, some embodiments ofthe present invention use an RNA polymerase that lacks helicase-like activity.
  • some embodiments ofthe present invention use an N4 bacteriophage-derived mini-vRNAP ((PCT Publication No. WO 02/095002 A2) that lacks helicase-like activity.
  • Mini-vRNAP enzymes use single-sfranded DNA templates and are unable to unwind or transcribe double-sfranded DNA.
  • Mini-vRNAP enzymes require EcoSSB Protein to displace the RNA product from the RNA:DNA hybrid obtained from in vitro transcription of linear templates (Davidova, ⁇ K and Rothman-Denes, LB, Proc. Natl. Acad. Sci. USA, 100: 9250-9255, 2003).
  • the present invention also comprises other embodiments that use target probes that comprise a single-sfranded pseudopromoter or synthetic promoter that is obtained for an RNA polymerase such, but not limited to E. coli RNAP or T7 RNAP.
  • An anti-sense promoter oligo is not needed in these embodiments, which use a single-sfranded pseudopromoter or synthetic promoter obtained as described by Ohmichi et al. (Proc. Natl. Acad. Sci. USA, 99: 54-59, 2002).
  • bipartite target probes that lack a transcription promoter sequence are used to generate circular ssDNA transcription substrates for rolling circle transcription using an RNA polymerase such as but not limited to an E. coli or T7-type RN polymerase.
  • JP4304900 and JP4262799 disclosed use of phi29 DNA polymerase to replicate a circular ligation product * it was since shown that ligation of a similar probe when annealed to a target sequence created a circular DNA molecule catenated to the target nucleic acid comprising the target sequence and that rolling circle replication by phi 29 DNA polymerase was limited if the 3'-end o the target nucleic acid was more than about 150-200 bases from the target sequence (Nilsson, M. et al., Science, 265:2085- 2088, 1994; Baner, J. et al., Nucleic Acids Research, 26: 5073-5078, 1998). Since this was not known by Toshiya et al.
  • the present invention comprises steps to avoid problems due to catenation ofthe transcription substrate on the target nucleic acid.
  • Patent Nos. JP4304900 and JP4262799 did not appear to have been pursued.
  • the present invention discloses a variety of processes and methods that overcome these problems.
  • amplifying a target or “amplifying a target nucleic acid” or “amplifying a target nucleic acid sequence” or “amplifying a target sequence” herein mean increasing the number of copies of that portion ofthe sequence of a target nucleic acid for which a complementary sequence is present in a target probe ofthe invention, including, but not limited to, a target-complementary sequence that is present in a target probe that also comprises a sequence for a transcription promoter for an RNA polymerase.
  • An "amplified target” or an “amplified target sequence” comprises only that portion ofthe sequence of a target nucleic acid for which a complementary sequence is present in a target probe ofthe invention.
  • amplifying a target or “amplifying a target nucleic acid” or “amplifying a target nucleic acid sequence” or “amplifying a target sequence” herein is not intended to imply that all ofthe sequence of a target nucleic acid is amplified. The use of these terms is also not intended to imply that the amplification of that portion ofthe sequence of a target nucleic acid for which a complementary sequence is present in a target probe ofthe invention is actually directly observed or detected in a method or assay ofthe invention.
  • the invention comprises embodiments in which the amplified target sequence is directly detected, such as, but not limited to, embodiments in which the target sequence is detected by measuring a fluorescent signal following annealing of a transcript-complementary detection probe such as, but not limited to a molecular beacon.
  • the invention also comprises embodiments in which the amplified target sequence is detected only indirectly by generation of another signal, such as, but not limited to, embodiments in which a signal is generated as a result of transcription of another DNA sequence that is covalently attached to a target-complementary sequence and that is transcribed along with a target-complementary sequence.
  • the amplification of a target sequence is detected by detecting a substrate for Q-beta replicase.
  • the substrate is replicated by Q-beta replicase using replication conditions well known in the art following synthesis of said RNA subsfrate by transcription of a signal sequence portion of a target probe that encodes said Q-beta subsfrate.
  • the term "amplification signal" as used herein is intended to describe the output or result of any method, whether direct or indirect, for detecting if amplification of a target sequence has occurred.
  • an amplification signal can comprise a fluorescent signal that results from annealing of a molecular beacon to an RNA transcript that is complementary to a target probe, or an amplification signal can comprise a Q-beta substrate that is replicated by Q- beta replicase following transcription of a DNA portion of a target probe that encodes said Q- beta substrate.
  • the invention comprises any signal sequence and any detection method that detects target-dependent franscription of a target sequence or a signal sequence.
  • RNA-dependent DNA polymerase is an enzyme that synthesizes a complementary DNA copy ("cDNA") from an RNA template. All known reverse franscriptases also have the ability to make a complementary DNA copy from a DNA template; thus, they are both RNA- and DNA-dependent DNA polymerases.
  • a primer is required to initiate synthesis with both RNA and DNA templates.
  • reverse transcriptases that can be used in methods ofthe present invention include, but are not limited to, AMV reverse transcriptase, MMLV reverse franscriptase, Tth DNA polymerase, rBst DNA polymerase large fragment, also called IsoThermTM DNA Polymerase (EPICENTRE Technologies, Madison, WI, USA), and BcaBESTTM DNA polymerase (Takara Shuzo Co, Kyoto, Japan).
  • a mutant form of a reverse franscriptase such as, but not limited to, an AMV or MMLV reverse franscriptase that lacks RNase H activity can be used.
  • a wild-type enzyme is preferred.
  • a separate RNase H enzyme such as but not limited to, E. coli RNase H or HybridaseTM Thermostable RNase H (EPICENTRE Technologies, Madison, WI 53713, USA) can also be used in reverse transcription reactions.
  • MMLV reverse franscriptase wild-type, RNase H-positive
  • IsoThermTM DNA polymerase or AMV reverse transcriptase can be used.
  • the processes ofthe invention include conducting experiments to determine the effects on amplification of RNase H activity of a reverse franscriptase and/or separate RNase H enzyme(s) used, including, but not limited to, AMV reverse transcriptase, IsoTherm DNA polymerase, and both RNase H-plus and RNase H-minus MMLV reverse transcriptase, and E. coli RNase H or thermostable RNase H enzymes that are stable for more than 10 minutes at 70° C (U.S. Patents Nos.
  • 5,399,491 discloses information related to the effects of adding different amounts of a separate RNase H enzyme to transcription-mediated amplification assays that used T7 RNAP and dsDNA templates and either MMLV or AMV reverse transcriptase, which information is useful in suggesting how to vary and evaluate reaction conditions related to use of reverse transcriptases and RNase H enzymes in methods and assays ofthe present invention.
  • a DNA polymerase for use in an embodiment ofthe invention that comprises rolling circle replication can be readily determined.
  • the ability of a polymerase to carry out rolling circle replication can be determined by using the polymerase in a rolling circle replication assay as described by Fire and Xu (Proc. Natl. Acad. Sci. USA, 92: 4641-4645, 1995), inco ⁇ orated herein by reference.
  • a DNA polymerase be a strand displacing DNA polymerase and lack a 5'-to-3' exonuclease activity for strand displacement polymerization reactions using both linear or circular templates since a 5'-to-3' exonuclease activity, if present, might result in the destruction ofthe synthesized strand. It is also preferred that DNA polymerases for use in the disclosed strand displacement synthesis methods are highly processive. The ability of a DNA polymerase to strand-displace can vary with reaction conditions, in addition to the particular enzyme used. Sfrand displacement and DNA polymerase processivity can also be assayed using methods described in Kong et al. (J. Biol.
  • Preferred strand displacing DNA polymerases ofthe invention are RepliPHF M phi29 DNA polymerase (EPICENTRE Technologies, Madison, WI, USA), phi29 DNA polymerase, rBst DNA polymerase large fragment (also called IsoThermTM DNA polymerase (EPICENTRE Technologies, Madison, WI, USA), BcaBESTTM DNA polymerase (Takara Shuzo Co., Kyoto, Japan), and SequiThermTM DNA polymerase (EPICENTRE Technologies, Madison, WI, USA).
  • strand-displacing DNA polymerases which can be used include, but are not limited to phage M2 DNA polymerase (Matsumoto et al., Gene, 84: 247, 1989), phage phi PRD1 DNA polymerase (Jung et al., Proc. Natl. Acad. Sci. USA, 84: 8287, 1987), VENT® DNA polymerase (Kong et al., J. Biol. Chem. 268: 1965-1975, 1993), Klenow fragment of DNA polymerase I (Jacobsen et ah, Eur. J. Biochem.
  • the amount of strand-displacing DNA polymerase in the reaction be as high as possible without inhibiting the reaction.
  • Re ⁇ liPHF M phi29 DNA Polymerase can be used at about 0.05 microgram to about one microgram of protein in a 20-microliter reaction and IsoThermTM DNA Polymerase can be used at about 50 units to about 300 units in a 50-microliter reaction.
  • strand displacement can be facilitated for some DNA polymerases through the use of a strand displacement factor, such as a helicase. It is considered that any DNA polymerase that can perform rolling circle replication in the presence of a strand displacement factor is suitable for use in embodiments ofthe invention that comprise rolling circle replication, even if the DNA polymerase does not perform rolling circle replication in the absence of such a factor.
  • Sfrand displacement factors useful in rolling circle replication include, but are not limited to, BMRF1 polymerase accessory subunit (Tsurumi et al., J. Virology, 67: 7648-7653, 1993), adenovirus DNA-binding protein (Zijderveld and van der Vliet, J. Virology, 68: 1158-1164, 1994), he ⁇ es simplex viral protein ICP8 (Boehmer and Lehman, J. Virology, 67: 711-715, 1993); Skaliter and Lehman, Proc. Natl. Acad. Sci. USA, 91: 10,665-10,669, 1994), single-stranded DNA binding proteins (SSB; Rigler and Romano, J.
  • BMRF1 polymerase accessory subunit Tesurumi et al., J. Virology, 67: 7648-7653, 1993
  • adenovirus DNA-binding protein Zijderveld and van der Vliet
  • Still another embodiment ofthe invention comprises a composition of promoter target probe that is immobilized or attached to a solid support.
  • the promoter target probe can have a moiety, such as but not limited to a biotin moiety on or near to its 3'-end that permits attachment ofthe promoter target probe to a solid support after annealing to the target sequence and ligation to an adjacently annealed target probe, such as a signal target probe or a simple target probe.
  • the ligation product obtained by ligating the promoter target probe to the adjacently annealed target probe on the target sequence generates a transcription subsfrate ofthe present invention, whether the promoter target probe is attached to a solid support or has a biotin or other moiety that permits attachment to a solid support.
  • One reason to attach the promoter target probe to a solid support after ligating to an adjacently annealed target probe on a target sequence is that solution hybridization is generally more efficient than hybridization on a surface.
  • the complexing of an anti-sense promoter oligo with the ligation product can be before or after the ligation product is attached to the solid support.
  • a promoter target probe that is to be attached to a solid support can comprise a biotin moiety at or near its 3 '-end, in which case, the promoter target probe can be attached to a solid support that is covalently or non-covalently joined to an avidin or sfreptavidin moiety using any ofthe variety of joining methods known in the art.
  • the promoter target probe is attached to a solid support prior to annealing to the target sequence and ligating to one or more other target probes or is attached to the solid support after annealing to the target sequence and being ligated to one or more other target probes and/or complexing with an anti-sense promoter oligo
  • the promoter target probe or the resulting ligation product is immobilized on the solid support at or near its 3 '-end and is at a sufficient distance from the surface ofthe solid support so that the promoter in double-sfranded form can bind a cognate RNA polymerase and initiate transcription therefrom under suitable transcription conditions.
  • a biotin may be attached to the promoter target probe, for example, but without limitation, by using a ribonucleoside triphosphate that is derivatized with biotin.
  • a ribonucleoside triphosphate that is derivatized with biotin.
  • Exemplary methods for making derivatized nucleoside triphosphates are disclosed in detail in Rashtchian et al., "Nonradioactive Labeling and Detection of Biomolecules," C. Kessler, Ed., Springer- Verlag, New York, pp. 70-84, 1992, herein inco ⁇ orated by reference.
  • the solid support has a chemical composition and structure so that it does not non-specifically bind nucleic acid from a sample or that comprises a composition ofthe invention, such as, but not limited to a sense promoter primer.
  • the solid support has a chemical composition and structure so that it does not non-specifically bind enzymes, co-factors or other substances in reactions comprising methods ofthe invention.
  • solid supports can comprise dipsticks, membranes, such as nitrocellulose or nylon membranes, beads, chips or slides used for making arrays or microarrays, and the like.
  • a circular franscription subsfrate is obtained using monopartite target probes rather than a bipartite target probe.
  • the monopartite target probes anneal to the target sequence and are ligated in the presence of a target sequence to form a linear ligation product as described previously.
  • the linear ligation product is denatured from the target sequence and subsequently circularized by ligation of its 3'-end to its 5'-end.
  • the 5'-end ofthe linear ligation product has a 5'-phosphate group or is phosphorylated using a kinase, such as but not limited to T4 polynucleotide kinase, in the presence of ATP.
  • a kinase such as but not limited to T4 polynucleotide kinase
  • This 5 '-phosphorylated linear ligation product is then complexed with a ligation splint oligo that has ends that are complementary to the 3'- end and the 5 '-end ofthe linear ligation product and the ends are ligated under ligation conditions with a ligase that has little or no activity in ligating blunt ends and that is substantially more active in ligating ends that are adjacent when annealed to a contiguous complementary sequence than if the ends are not annealed to the complementary sequence, such as but not limited to Ampligase® DNA Ligase (EPICENTRE Technologies, Madison, WI).
  • a ligation splint and a ligase such as Ampligase® DNA Ligase
  • a ligase such as Ampligase® DNA Ligase
  • the same ligase is used both for ligation ofthe target probes annealed to the target sequence and for subsequent ligation ofthe 3'-end to the 5'-end ofthe ligation product using a ligation splint. After annealing an anti- sense promoter oligo to the circularized ligation product, a circular franscription substrate ofthe invention is obtained.
  • initiation of RNA synthesis with these circular ssDNAs occurs primarily with pppG, as is usually the case for promoter-initiated franscription using these RNA polymerases.
  • a linear precursor of a circular ssDNA is franscribed little or not at all under conditions in which the corresponding circular ssDNA is transcribed efficiently.
  • a "simple bipartite target probe" is used that lacks a known promoter sequence.
  • a "simple bipartite target probe” comprises a linear ssDNA precursor to a circular ssDNA molecule, wherein the 3 '-end portion and the 5 '-end portion of said linear ssDNA precursor comprise sequences that are complementary, respectively, to the most 5 '-portion and the most 3 '-portion of a target nucleic acid sequence.
  • a simple bipartite target probe of this embodiment of an assay or method ofthe invention comprises a linear ssDNA precursor of a circular ssDNA molecule, wherein said simple bipartite target probe is franscribed little or not at all, but said circular ssDNA molecule is an efficient template for rolling circle transcription by an RNA polymerase used in said assay or method.
  • annealing of said simple bipartite target probe to a target sequence and target sequence-dependent ligation of said simple bipartite target probe during a process of an assay or method ofthe invention yields a circular ssDNA molecule comprising a "simple circular transcription substrate" ofthe invention.
  • a circular ssDNA molecule such as, but not limited to, those reported by Daubendiek et al. and by Kool, is identified as an efficient substrate for rolling circle transcription.
  • Circular ssDNA molecules can be made as described (Prakash, G and Kool, E.T., J. Am. Chem. Soc, 114: 3523-3527, 1992; Wang, S. and Kool, E.T., Nucleic Acids Res., 22: 2326-2333, 1994; Kool, E.T. in US Patent Nos. 5,714,320; 6,077,668; 6,096,880; and 6,368,802 Bl, all of which are inco ⁇ orated herein in their entirety by reference).
  • one embodiment ofthe invention is a method for detecting a target nucleic acid sequence, the method comprising: (a) providing a simple bipartite target probe comprising linear single-sfranded DNA (ssDNA) that lacks a sequence for a known promoter for an RNA polymerase, the simple bipartite target probe comprising two target-complementary sequences that are not joined to each other, wherein the 5 '-end of a first target-complementary sequence is complementary to the 5'-end ofthe target nucleic acid sequence, and wherein the 3'-end of a second target-complementary sequence is complementary to the 3 '-end ofthe target nucleic acid sequence, wherein said simple bipartite target probe is franscribed little or not at all by an RNA polymerase under conditions in which a circular ssDNA obtained by
  • the ligase has little or no activity in ligating blunt ends and is substantially more active in ligating ends that are adjacent when annealed to two contiguous regions of a target sequence compared to ends that are not annealed to the target sequence.
  • One suitable ligase that can be used is Ampligase® Thermostable DNA Ligase (EPICENTRE Technologies, Madison, WI).
  • the RNA polymerase comprises an RNA polymerase chosen from among a T7 RNAP, a T3 RNAP, an SP6 RNAP or another T7- like RNA polymerase, including mutant forms thereof, or E. coli RNA polymerase or Thermus thermophilus RNA polymerase.
  • the target sequence comprises a target nucleic acid in a sample, whereas in other embodiments the target sequence comprises a target sequence tag that is joined to an analyte-binding substance that binds an analyte in the sample.
  • the method is used to detect a single-nucleotide polymo ⁇ hism (SNP) or mutation, in which case the 5'-nucleotide ofthe first target-complementary sequence or the 3'- end ofthe second target-complementary sequence of said simple bipartite target probe is complementary to the intended target nucleotide ofthe target sequence, and ligation only occurs when the ends of both target-complementary sequences are adjacently annealed on the target sequence, including the target nucleotide, under the stringent ligation conditions ofthe assay or method.
  • the target sequence is preferably less than about 150 to about 200 nucleotides from the 3'-end ofthe target nucleic acid or target sequence tag.
  • the target sequence is greater than about 150 to about 200 nucleotides from the 3'-end ofthe target nucleic acid or target sequence tag comprising the target sequence
  • one or more additional steps is used in order to release the catenated circular ligation product from the target sequence prior to franscription, as described elsewhere herein.
  • the circular transcription subsfrate that is franscribed remains catenated to a target nucleic acid.
  • a simple bipartite target probe can also serve as a target sequence amplification probe (or TSA probe) that is used as described elsewhere herein to obtain additional target sequences for target-dependent ligation ofthe simple bipartite target probe to make additional simple circular franscription subsfrates.
  • TSA probe target sequence amplification probe
  • a primer that anneals to the sequence between the target-complementary portions is provided to prime rolling circle transcription of a TSA circle that results from ligation of a TSA probe annealed to a target sequence.
  • Simple bipartite probes can also be used in other embodiments for secondary amplification of an RNA transcription product or of a reverse transcription product derived therefrom.
  • a simple bipartite secondary amplification probe comprising end sequences that are complementary to other sequences than target sequences are used and the simple bipartite secondary amplification probe is annealed and ligated on either an RNA template resulting from franscription of a target-dependent transcription subsfrate or on a cDNA reverse franscription product derived from said RNA franscription product.
  • the simple bipartite secondary amplification probe can be annealed and ligated on an RNA transcript or its cDNA product conesponding to a signal sequence portion of a franscription subsfrate.
  • This embodiment is better understood following a complete reading ofthe description ofthe invention herein
  • the present invention comprises methods, compositions and kits for detecting one or multiple specific target sequences in a sample by target-dependent transcription.
  • Figure 3 shows one basic embodiment of a method ofthe present invention. This embodiment uses a bipartite target probe.
  • a bipartite target probe is a linear single-sfranded DNA molecule that has sequences on both ends ofthe probe that are complementary to different portions of a target sequence.
  • the target-complementary sequences ofthe bipartite target probe are contiguous or adjacent or abut to each other when annealed to the target sequence.
  • the sequence at the 5 '-end ofthe bipartite target probe preferably has a 5'- phosphate group or is phosphorylated by a polynucleotide kinase during the course of a method ofthe invention.
  • the 5 '-portion ofthe bipartite target probe also has sequence for a sense promoter sequence for a functional promoter for a DNA-dependent RNA polymerase, which upon complexing with an anti-sense promoter oligo, can bind to this double-sfranded promoter and initiate transcription of RNA therefrom in a 5'-to-3' direction using single-sfranded DNA that is 5 '-of and covalently linked to the promoter as a template.
  • the promoter is oriented within the single-stranded DNA ofthe bipartite target probe 3 '-of the target-complementary sequence at the 5 '-end ofthe 5 '-portion.
  • the sequence at the 3 '-end ofthe bipartite target probe preferably has a 3'-hydroxyl group.
  • a bipartite target probe anneals to the target sequence under hybridization conditions, wherein the 5 '-phosphorylated end ofthe bipartite target probe is adjacent to its 3'-hydroxyl end.
  • the ends ofthe bipartite target probe are ligated under ligation conditions by contacting the target-complementary ends annealed to a target sequence with a ligase that has little or no activity in ligating free ends that are not annealed to a complementary sequence but is active in joining a 5 '-phosphorylated end to a 3'-hydroxylated end when the ends are adjacent when annealed to a complementary DNA sequence.
  • Ligation ofthe ends ofthe bipartite target probe generates a "circular transcription substrate," meaning a circular single-sfranded DNA molecule that is a template for franscription by an RNA polymerase that recognizes a promoter sequence in said circular transcription substrate.
  • one embodiment of the present invention comprises a method for detecting a target sequence, said method comprising: [00257] a. providing a bipartite target probe, wherein said bipartite target probe comprises a linear single-stranded DNA comprising two separate target-complementary end portions that are complementary to a contiguous target sequence;
  • c ligating said bipartite target probe annealed to said target sequence under ligation conditions with a ligase, wherein said ligase has little or no activity in ligating blunt ends and is substantially more active in ligating said ends of said bipartite target probe if said ends are adjacent when annealed to two contiguous regions of a target sequence than if said ends are not annealed to said target sequence, so as to obtain a circular ssDNA ligation product; [00260] d.
  • the anti-sense promoter oligo is attached to a solid support.
  • the anti-sense promoter oligo comprises a moiety, such as, but not limited to a biotin moiety that permits binding ofthe anti-sense promoter oligo to a solid support after annealing to the ligation product to obtain a transcription subsfrate.
  • the invention also comprises a method for amplifying a target sequence, said method comprising:
  • ssDNA linear single-sfranded DNA
  • [00268] c. ligating said bipartite target probe annealed to said target sequence under ligation conditions with a ligase, wherein said ligase has little or no activity in ligating blunt ends and is substantially more active in ligating said ends of said bipartite target probe if said ends are adjacent when annealed to two contiguous regions of a target sequence than if said ends are not annealed to said target sequence, so as to obtain a circular ssDNA ligation product;
  • Figure 4 shows an embodiment of a method or assay ofthe invention that is similar to the embodiment shown in Figure 3 except that the bipartite target probe used in the method shown in Figure 4 does not have a franscription termination sequence and franscription ofthe circular franscription substrate resulting therefrom generates a franscription product comprising a transcription product multimer by rolling circle transcription.
  • one embodiment ofthe present invention comprises a method for detecting a target sequence, said method comprising:
  • bipartite target probe comprising a linear single-stranded DNA comprising two separate target-complementary end portions that are complementary to a contiguous target sequence
  • [00277] c ligating said bipartite target probe annealed to said target sequence under ligation conditions with a ligase, wherein said ligase has little or no activity in ligating blunt ends and is substantially more active in ligating said ends of said bipartite target probe if said ends are adjacent when annealed to two contiguous regions of a target sequence than if said ends are not annealed to said target sequence, so as to obtain a circular ssDNA ligation product;
  • f contacting said circular franscription substrate with an RNA polymerase under rolling circle transcription conditions so as to synthesize transcription product multimers, wherein a transcription product multimer comprises multiple tandem copies of an oligomer that is complementary to one copy of said circular franscription substrate;
  • the target sequence is less than about 150 to about 200 nucleotides from the 3 '-end ofthe target nucleic acid or target sequence tag. If the target sequence is greater than about 150 to about 200 nucleotides from the 3'-end ofthe target nucleic acid or target sequence tag comprising the target sequence, then one or more additional steps (as described elsewhere herein) is required in order to release the catenated circular ligation product from the target sequence prior to transcription. In other embodiments, the circular transcription substrate that is transcribed remains catenated to a target nucleic acid.
  • the anti-sense promoter oligo is attached to a solid support.
  • the anti-sense promoter oligo comprises a moiety, such as, but not limited to a biotin moiety that permits binding ofthe anti-sense promoter oligo to a solid support after annealing to the ligation product to obtain a transcription substrate.
  • Other embodiments of the present invention as shown in Figure 5, comprise compositions, methods and kits for detecting one or multiple specific target sequences in a sample by coupled target-dependent rolling circle replication (RCR) and rolling circle transcription (RCT).
  • bipartite target probes to generate circular franscription substrates as shown in either Figure 3 or Figure 4, which result in circular transcription substrate either with a transcription terminator or lacking a transcription terminator, respectively. If the circular transcription subsfrate lacks a franscription terminator sequence, transcription comprises rolling circle transcription as described elsewhere herein.
  • these embodiments also use a "bipartite target sequence amplification probe,” which is also referred to as a "bipartite TSA probe” or simply as a "TSA probe” herein.
  • a TSA probe is a linear single-stranded DNA molecule that comprises two target-complementary sequences that are connected by an intervening sequence that is not complementary to the target sequence.
  • the target-complementary portions on the ends are complementary to different portions of a target sequence in a target nucleic acid or a target sequence tag of an analyte-binding substance.
  • Each ofthe 5' and 3' target-complementary sequences in a TSA probe for a particular assay or method is identical to the corresponding target-complementary sequence at the 5'-end or the 3'-end of a bipartite target probe used in the assay or method. That is, the 5'-end of a TSA probe anneals to the same nucleotides in the target sequence as the 5 '-end ofthe corresponding bipartite target probe that is used to obtain a circular franscription substrate and similarly, the 3 '-end ofthe TSA probe anneals to the same nucleotides ofthe target sequence as the 3'-end ofthe bipartite target probe.
  • the target-complementary sequences ofthe TSA probe are adjacent to each other when annealed to the target sequence in exactly the same manner as described previously for bipartite target probes.
  • the sequence at the 5'-end ofthe TSA probe preferably has a 5'- phosphate group or is phosphorylated by a polynucleotide kinase during the course of a method ofthe invention and the sequence at the 3 '-end of a TSA probe preferably has a 3'-hydroxyl group.
  • the adjacent target- complementary sequences of a TSA probe are ligated in a method ofthe invention with a ligase that has little or no activity in ligating blunt ends and that is substantially more active in ligating said ends that are adjacent when annealed to two contiguous regions of a target sequence than if said ends are not so annealed.
  • Ligation of a TSA probe results in formation of a "TSA circle," which, upon annealing to a primer, is a substrate for rolling circle replication.
  • TSA circle which, upon annealing to a primer, is a substrate for rolling circle replication.
  • the sequence and nucleotide composition ofthe intervening sequence can vary, but it should comprise a sequence of sufficient length and sequence specificity to provide a primer-binding site for specific priming by a primer for rolling circle replication.
  • the intervening sequence should also be of sufficient length to permit the target-complementary sequences ofthe TSA probe to anneal to the target sequence with specificity.
  • the length ofthe intervening sequence should be optimized to obtain the optimal target-dependent ligation efficiency with the ligase and the maximum rolling circle replication rate and maximum end-point level of RCR product with the strand-displacing DNA polymerase under the assay conditions used.
  • a bipartite target probe could also be used as a TSA probe, it is preferable that the TSA probe is not a bipartite target probe.
  • a TSA probe does not have a franscription promoter sequence, a transcription termination sequence, or a signal sequence, and preferably the primer-binding site in a TSA probe for a strand-displacing DNA polymerase primer used for rolling circle replication is not present in the corresponding bipartite target probe.
  • the lack of a promoter sequence in the TSA probe or the resulting TSA circle permits maximum rolling replication because there is no promoter to bind an RNA polymerase or initiate transcription.
  • the lack of a primer-binding site for priming by a strand- displacing DNA polymerase on the bipartite target probe or the resulting circular transcription substrate permits maximum transcription because there is not site for priming a competitive rolling circle franscription reaction.
  • a TSA probe anneals to the target sequence under hybridization conditions, wherein the 5 '-phosphorylated end ofthe TSA probe is adjacent to its 3'-hydroxyl end. Then, the ends ofthe TSA probe are ligated under ligation conditions by contacting the target-complementary ends annealed to a target sequence with a ligase that has little or no activity in ligating free ends that are not annealed to a complementary sequence but is active in joining a 5 '-phosphorylated end to a 3'-hydroxylated end when the ends are adjacent when annealed to a complementary DNA sequence. Ligation of the ends ofthe TSA probe generates a TSA circle.
  • the adjacent 5'-phosphorylated end and the 3'-hydroxyl end o the bipartite target probes annealed to the tandem target sequences ofthe rolling circle replication products are ligated by the ligase under ligation conditions, thereby generating a ligation product which, upon annealing of an anti-sense promoter oligo results in a circular franscription subsfrate.
  • Transcription products are obtained by contacting the circular transcription subsfrates with an RNA polymerase that can bind the double-stranded promoter and initiate franscription therefrom, and the franscription products are obtained or detected by a suitable means.
  • one embodiment ofthe present invention comprises a method for obtaining a franscription product complementary to a target nucleic acid sequence (target sequence or target), said method comprising:
  • TSA probe target sequence amplification probe
  • TSA probe comprises a linear single-stranded DNA comprising two end portions that are not joined, which end portions are connected by an intervening sequence, wherein the 5 '-end target- complementary sequence is complementary to the 5' -end o he target sequence, and wherem the 3'-end target-complementary sequence is complementary to the 3'-end ofthe target sequence, and wherein joining ofthe ends of said TSA probe forms a TSA circle; [00290] b. contacting the TSA probe to the target sequence and incubating under hybridization conditions, wherein the target-complementary sequences anneal adjacently to the target sequence;
  • target probes comprising linear single-sfranded DNA
  • the target probes comprising at least two target-complementary sequences that are not joined to each other, wherein the 5 '-end of a first target-complementary sequence is complementary to the 5 '-end of the target sequence, and wherein the 3 '-end of a second target-complementary sequence is complementary to the 3 '-end ofthe target sequence, and wherein the 3 '-end ofthe first target- complementary sequence is joined to the 5 '-end of a sense promoter sequence for an RNA polymerase;
  • k contacting the transcription substrate with an RNA polymerase that can bind the promoter and incubating under transcription conditions to obtain a transcription product; and [00299] 1. detecting the franscription product, wherein said transcription product indicates the presence of said target sequence.
  • the ligase has little or no activity in ligating blunt ends and is substantially more active in ligating ends that are adjacent when annealed to two contiguous regions of a target sequence compared to ends that are not annealed to the target sequence.
  • One suitable ligase that can be used is Ampligase® Thermostable DNA Ligase (EPICENTRE Technologies, Madison, WI).
  • a preferred strand-displacing DNA polymerase that can be used is IsoThermTM DNA Polymerase (EPICENTRE Technologies, Madison, WI).
  • RNA polymerases are T7 RNAP, T3 RNAP, SP6 RNAP or another T7-like RNA polymerase or a mutant form of one of these T7- like RNA polymerases.
  • AmpliScribe T7-FlashTM Transcription Kit is used for in vitro franscription ofthe transcription subsfrate (EPICENTRE Technologies, Madison, WI).
  • the target probes comprise monopartite target probes comprising a promoter target probe and a signal target probe and/or one or more simple target probes. In other embodiments, the target probes comprise a bipartite target probe and optionally, one or more simple target probes.
  • one embodiment ofthe present invention comprises a method for obtaining a transcription product complementary to a target nucleic acid sequence (target or target sequence), said method comprising:
  • TSA probe target sequence amplification probe
  • TSA probe comprises a linear single-stranded DNA comprising two end portions that are not joined, which end portions are connected by an intervening sequence, wherein the 5 '-end target- complementary sequence is complementary to the 5 '-end ofthe target sequence, and wherein the 3 '-end target-complementary sequence is complementary to the 3 '-end ofthe target sequence, and wherein joining ofthe ends of said TSA probe forms a TSA circle; [00304] b. providing a primer that is complementary to the intervening sequence of said
  • bipartite target probe comprising a linear ssDNA comprising two end portions that are not joined, wherein the 5 '-end of a first target-complementary sequence is complementary to the 5 '-end ofthe target nucleic acid sequence, and wherein the 3 '-end of a second target-complementary sequence is complementary to the 3 '-end ofthe target nucleic acid sequence, and wherein the 3 '-end ofthe first target- complementary sequence is joined to the 5 '-end of a sense promoter sequence for an RNA polymerase;
  • [00312] j annealing an anti-sense promoter oligo to said circular ssDNA ligation product to obtain a circular transcription subsfrate; [00313] k. obtaining said circular franscription substrate, wherein said circular transcription substrate comprises a sequence that is complementary to said target sequence; [00314] 1. contacting said circular franscription subsfrate with an RNA polymerase under transcription conditions so as to obtain a transcription product that is complementary to said circular transcription subsfrate; and
  • the ligase has little or no activity in ligating blunt ends and is substantially more active in ligating ends that are adjacent when annealed to two contiguous regions of a target sequence compared to ends that are not annealed to the target sequence.
  • One suitable ligase that can be used is Ampligase® Thermostable DNA Ligase (EPICENTRE Technologies, Madison, WI).
  • a preferred strand-displacing DNA polymerase that can be used is IsoThermTM DNA Polymerase (EPICENTRE Technologies, Madison, WI).
  • RNA polymerases are T7 RNAP, T3 RNAP, SP6 RNAP or another T7-like RNA polymerase or a mutant form of one of these T7- like RNA polymerases.
  • AmpliScribe T7-FlashTM Transcription Kit is used for in vitro transcription ofthe franscription substrate (EPICENTRE Technologies, Madison, WI).
  • the anti-sense promoter oligo is attached to a solid support.
  • the anti-sense promoter oligo comprises a moiety, such as, but not limited to a biotin moiety that permits binding ofthe anti-sense promoter oligo to a solid support after annealing to the ligation product to obtain a franscription substrate.
  • the target sequence comprises a target nucleic acid in a sample, whereas in other embodiments the target sequence comprises a target sequence tag that is joined to an analyte- binding substance that binds an analyte in the sample.
  • the TSA circle that is replicated remains catenated to a target nucleic acid or target sequence tag.
  • the target sequence is less than about 150 to about 200 nucleotides from the 3'-end of the target nucleic acid or target sequence tag.
  • the target sequence is greater than about 150 to about 200 nucleotides from the 3 '-end ofthe target nucleic acid or target sequence tag, then one or more additional steps is used in order to release the catenated TSA circles from the target sequence prior to rolling circle replication, as described elsewhere herein.
  • one or more additional steps can be used in order to release the catenated circular ssDNA ligation products that result from ligation of bipartite target probes that are annealed to target sequences in the rolling circle replication product more than about 150 nucleotides to about 200 nucleotides from the 3'-end of to the rolling circle replication product.
  • rolling circle replication is carried out using a ratio of dUTP to dTTP that results in inco ⁇ oration of a dUMP residue about every 100-400 nucleotides and a composition comprising uracil-N-glycosylase and endonuclease TV is used to release catenated DNA molecules that are ligated on the linear rolling circle replication product following annealing of bipartite target probes to the replicated target sequences.
  • Figure 6 shows one aspect of another embodiment of a method ofthe present invention.
  • This embodiment also uses a bipartite target probe that is similar to a bipartite target probe used in the method shown in Figure 3, except that the target-complementary sequences of the bipartite target probe used in the embodiment shown in Figure 6 are not contiguous or adjacent to each other when annealed to the target sequence. Rather, the target-complementary sequences of a bipartite target probe of this embodiment are separated from each other when they are annealed to the target sequence.
  • the gap between the two target-complementary sequences can comprise from about four nucleotides to about 1000 nucleotides or more.
  • the gap in this embodiment o the invention comprises from about six nucleotides to about 100 nucleotides, and most preferably, the gap comprises from about six nucleotides to about 25 nucleotides.
  • the 5 '-end of the bipartite target probe in the embodiment in Figure 6 preferably has a 5 '-phosphate group or is phosphorylated by a polynucleotide kinase during the course of a method ofthe invention, and the 3 '-end preferably has a 3'-hydroxyl group.
  • the 5 '-portion ofthe bipartite target probe in the embodiment of Figure 6 has sequence for a sense franscription promoter which, upon annealing to an anti-sense promoter oligo, is a functional promoter for a DNA-dependent RNA polymerase that can bind this double-sfranded promoter and initiate franscription therefrom in a 5'-to-3' direction, wherein the sense promoter sequence is joined to the 3'-end ofthe target- complementary sequence that anneals to the 5'-end ofthe target sequence. That is, the sense promoter is oriented within the single-stranded DNA of a bipartite target probe 3 '-of the target- complementary sequence at the 5'-end ofthe 5'-portion.
  • the gap between the target-complementary sequences of a bipartite target probe annealed to a target sequence is filled by also annealing one or more simple target probes comprising target- complementary sequences that anneal to the target sequence between portions ofthe target to which the target-complementary sequences ofthe bipartite target probe anneal.
  • the simple target probes used anneal to the target sequence so as to fill the gap completely so as to abut with or to be contiguous with each other and with the target-complementary sequences ofthe bipartite target probe.
  • All 5 '-ends of simple target probes and ofthe bipartite target probe have a 5'-phosphate group and all 3'-ends have hydroxyl groups.
  • ligation ofthe bipartite target probe and simple target probes that are annealed to a target sequence with a ligase which ligase has little or no activity in ligating free ends that are not annealed to a complementary sequence but is active in joining a 5 '-phosphorylated end to an adjacent 3'-hydroxylated end when the ends are annealed to a complementary DNA sequence, generates a circular ligation product, which upon annealing to an anti-sense promoter oligo, generates a circular transcription substrate.
  • Transcription ofthe circular transcription substrate results in synthesis of franscription product that is complementary to the circular transcription subsfrate and that can be used to detect the presence ofthe target sequence.
  • one embodiment ofthe present invention comprises a method for detecting a target sequence, said method comprising: [00321 ] a. providing a bipartite target probe, wherein said bipartite target probe comprises a linear single-sfranded DNA comprising two end portions that are not joined and that are complementary to different non-contiguous 5'- and 3'- end portions ofthe target sequence; [00322] b.
  • said simple target probes are complementary to the target sequence so as to anneal to said target sequence in the gap between the target-complementary sequences of said bipartite target probe so as to completely fill said gap and so that each ofthe ends of said simple target probes are contiguous with an end of a simple target probe or with an end of said bipartite target probe;
  • Figure 7 shows another aspect of an embodiment of a method ofthe present invention that uses a bipartite target probe that comprises target-complementary sequences that are separated from each other when they are annealed to a target sequence.
  • the gap between the target-complementary sequences of a bipartite target probe annealed to a target sequence is filled by primer extension using a DNA polymerase and subsequently joined by ligation with a ligase if and only if both the 3'-end ofthe target probe that is annealed to the target sequence 3 '-of the gap and the target probe that is annealed to the target sequence 5 '-of the gap are complementary to and correctly basepaired with the target sequence.
  • the DNA polymerase will be unable to fill the gap by primer extension.
  • the 3'-end ofthe target probe that is 5'-of the gap is not annealed to the target sequence, then the 3'-end ofthe primer extension product will not be adjacent to a 5'-end on the target sequence and it will not be possible to join the 3'-end ofthe primer-extended target probe with the 5'- phosphorylated end ofthe target probe annealed 5'-of the gap.
  • the gap between the two target-complementary sequences can comprise from one nucleotide to about 1000 nucleotides or more. Although the invention is not limited to a particular distance between the target-complementary sequences when annealed to a target sequence, preferably the gap comprises from one nucleotide to about 100 nucleotides, and most preferably, the gap in most embodiments comprises from one nucleotide to about 25 nucleotides.
  • the 5'-end of a bipartite target probe in the embodiment in Figure 7 preferably has a 5'- phosphate group or is phosphorylated by a polynucleotide kinase during the course of a method ofthe invention, and the 3 '-end preferably has a 3 '-hydroxyl group.
  • the 5 '-portion ofthe bipartite target probe in the embodiment of Figure 7 has sense promoter sequence which, upon complexing with an anti-sense promoter oligo, forms a functional promoter for a DNA- dependent RNA polymerase that can bind to this double-sfranded promoter and initiate franscription therefrom in a 5'-to-3' direction using as a template a single-sfranded DNA that is 5 '-of and covalently linked to the promoter.
  • the sense promoter sequence is oriented within the single-sfranded DNA of a bipartite target probe 3 '-of the target-complementary sequence at the 5 '-end ofthe 5 '-portion.
  • the gap between the target- complementary sequences of a bipartite target probe annealed to a target sequence is filled by contacting the target sequence to which a bipartite target probe is annealed with a DNA polymerase under polymerization conditions.
  • the 5 '-phosphorylated end of a bipartite target probe annealed to a target sequence is joined to the 3 '-end ofthe DNA polymerase- extended 3 '-end of said bipartite target probe with a ligase, which ligase has little or no activity in ligating free ends that are not annealed to a complementary sequence but is active in joining a 5 '-phosphorylated end to an adjacent 3'-hydroxylated end when the ends are annealed to a complementary DNA sequence, generates a circular franscription subsfrate. Transcription ofthe circular franscription subsfrate results in synthesis of franscription product that is complementary to the circular transcription substrate and that can be used to detect the presence ofthe target sequence.
  • one embodiment ofthe present invention comprises a method for detecting a target sequence, said method comprising:
  • bipartite target probe comprising a linear single-sfranded DNA comprising two end portions that are complementary to different non-contiguous 5'- and 3 '-end portions of a target sequence;
  • b annealing said bipartite target probe to said target sequence under hybridization conditions;
  • the invention also comprises methods that use a combination of both one or more simple target probes and DNA polymerase extension in order to fill the gap so as to obtain adjacent target-complementary sequences prior to the ligation step.
  • a suitable non-sfrand-displacing DNA polymerase for filling a gap according to this embodiment ofthe invention is T4 DNA polymerase.
  • FIG. 8 shows a basic embodiment of a method that generates a linear transcription substrate. This embodiment uses only monopartite target probes.
  • a monopartite target probe is a single-sfranded DNA molecule that comprises only one target-complementary sequence, although a monopartite target probe can comprise other sequences that are not complementary to a target sequence.
  • a method ofthe invention that generates a linear transcription substrate always uses a monopartite target probe called a "promoter target probe.”
  • a "promoter target probe” has a 5 '-portion that is complementary to the most 5 '-portion of a target sequence.
  • the 3'-end of this 5 '-target-complementary portion is joined to the 5'-end of a sense promoter sequence, which upon complexing with an anti-sense promoter sequence, serves as a functional transcription promoter for a DNA-dependent RNA polymerase that can bind to this promoter and initiate transcription of RNA therefrom in a 5'-to-3' direction under transcription conditions using single-stranded DNA that is 5 '-of (with respect to the same sfrand) and covalently linked to the promoter as a template.
  • the sequence at the 5'-end ofthe promoter target probe preferably has a 5' -phosphate group or is phosphorylated by a polynucleotide kinase during the course of a method ofthe invention.
  • a “signal target probe” has a 3 '-portion and a 5 '-portion. At least the 3 '-end portion of a signal target probe comprises a sequence that is complementary to the most 3 '-portion of a target sequence. As shown in the embodiment in Figure 8, the 3'-end ofthe signal target probe has a 3'-hydroxyl group.
  • the 5'-portion of a signal target probe comprises a "signal sequence.”
  • a signal sequence is a sequence that is detectable in some way following its transcription during a method ofthe invention. The invention does not require the use of a signal target probe having a signal sequence.
  • a simple target probe could be used in an assay ofthe invention in place of a signal target probe.
  • the signal sequence can comprise any sequence that is detectable following transcription.
  • a signal sequence can comprise a sequence that is detectable using a molecular beacon as described by Tyagi et al. (U.S. Patents Nos. 5,925,517 and 6,103,476 of Tyagi et al. and 6,461,817 of Alland et al., all of which are inco ⁇ orated herein by reference).
  • a prefened signal sequence ofthe invention is a sequence that results in an additional amplification ofthe signal following its transcription, thus making the detection of a target sequence more sensitive.
  • the signal target probe used in the method shown in Figure 8 can be, for example, a signal sequence that encodes a subsfrate for Q-beta replicase (EPICENTRE Technologies, Madison, WI), which permits additional amplification ofthe signal by incubating the transcription product with Q-beta replicase under replication conditions.
  • many other signal sequences can be used in a signal target probe, all of which are inco ⁇ orated as part ofthe present invention.
  • one embodiment ofthe present invention comprises a method for detecting a target sequence, said method comprising: [00345] a. providing a promoter target probe comprising a linear single-sfranded DNA having a 5 '-end comprising a target-complementary sequence that is complementary to the most 5'-portion ofthe target sequence, the 3'-end of which target-complementary sequence is joined to 5'-end of a sense promoter sequence for an RNA polymerase;
  • the invention also comprises embodiments in which a DNA polymerase is used to fill the gap between a promoter target probe and a signal target probe, wherein said target probes are not adjacent when annealed to a target sequence.
  • the invention further comprises use of a combination of a promoter target probe, one or more simple target probes, a signal target probe and DNA polymerase extension ofthe 3 '-hydroxyl end of said signal target probe or of one or more simple target probes so that said target probes, including said DNA polymerase-extended target probes, completely fill the gap on a target sequence between the target-complementary portions of said promoter probe and said signal target probe.
  • any combination of simple target probes and DNA polymerase extension can be used to fill the gap between the target-complementary portions ofthe promoter probe and the signal target probe so as to obtain adjacent target-complementary sequences prior to the ligation step.
  • the invention also comprises methods for obtaining secondary or additional amplification by using the transcription products synthesized by transcription of a circular franscription substrate or a linear transcription subsfrate as a template for ligation ofthe same or different bipartite or monopartite target probes, thus generating additional circular transcription subsfrates or linear franscription subsfrates, respectively.
  • one embodiment of a method ofthe invention for obtaining secondary amplification uses bipartite target probes and two ligases -one ligase that can ligate target-complementary sequences of a bipartite target probe annealed to a DNA target sequence to form a first circular ligation product that, upon annealing of an anti-sense promoter oligo, results in a first circular franscription substrate, and one that can ligate the same target-complementary sequences of said bipartite target probe annealed to an RNA transcript resulting from transcription of said first circular transcription substrate.
  • one ligase that can be used in a method ofthe present invention for ligation of contiguous DNA molecules annealed to an RNA ligation template is T4 RNA ligase (EPICENTRE Technologies, Madison, WI, USA), as disclosed by Faruqi in U.S. Patent No. 6,368,801 Bl, which is inco ⁇ orated herein by reference.
  • the invention also comprises embodiments that use similar secondary amplification methods with two ligases using monopartite target probes and that generate linear transcription substrates.
  • the invention also comprises other embodiments of methods and assays wherein a second bipartite target probe is used that anneals to a sequence in the RNA transcript that is complementary to a signal sequence or an optional sequence ofthe first circular franscription substrate rather than annealing to the target sequence or the identical sequence in the RNA transcript.
  • the invention also comprises use of a reverse franscriptase process to obtain additional amplification of a target sequence and/or a signal sequence in an assay or method ofthe invention.
  • a reverse transcriptase process is shown in Figure 9. This example illustrates a number of aspects ofthe invention that result in improvements over the methods and assays ofthe prior art.
  • the first part ofthe assay or method in Figure 9 is similar to the embodiment shown in Figure 4.
  • a first circular franscription substrate is generated by ligation of a first bipartite target probe annealed to a target sequence in a sample, followed by annealing of an anti-sense promoter oligo. Then, in vitro transcription ofthe first circular franscription substrate amplifies the target sequence and the signal sequence, if present.
  • rolling circle transcription is used to synthesize RNA comprising multimeric copies of an RNA oligomer that is complementary to the first circular transcription subsfrate.
  • rolling circle franscription synthesizes RNA that has sequences that are complementary to the sense promoter sequence in a circular franscription substrate.
  • RNA franscription product from rolling circle transcription permits generation of additional single-sfranded franscription promoters that can initiate additional in vitro franscription reactions and thereby further amplify the target sequence and/or signal sequence.
  • one or more oligonucleotide primers anneal to the multimeric RNA transcription products and first-strand cDNA is synthesized by extension of said primers by a reverse transcriptase under reverse transcription reaction conditions.
  • a reverse transcriptase under reverse transcription reaction conditions.
  • only one reverse franscription primer is used that anneals to the same sequence in different repeated sites on the multimeric RNA.
  • the invention also comprises embodiments that use multiple reverse transcription primers, each of which is complementary to a different sequence of an RNA oligomer that is, in turn, complementary to a circular transcription subsfrate.
  • the sequence to which a reverse transcription primer anneals in an RNA multimer can also vary.
  • a reverse transcription primer anneals to a sequence in the RNA multimer in a region that is complementary to an optional sequence portion of a circular franscription subsfrate and that is 3 '-of a signal sequence-complementary sequence, if present.
  • the reverse franscription primer shown in Figure 9 anneals to the RNA multimer at a site that is 3 'of of a signal sequence-complementary sequence of each oligomer ofthe multimer.
  • the reverse transcription primer shown in Figure 9 has a 5 '-portion comprising a "tail" that is a sequence that is not complementary to the RNA transcript.
  • the use of a tail is optional and is not required for methods and assays ofthe invention. As discussed below, a tail may be useful for embodiments that use a novel strand-displacement reverse transcription process ofthe present invention.
  • the first-sfrand cDNA is available in the reaction mixture for at least two subsequent functions.
  • the first-sfrand cDNA has a sense sequence for a franscription promoter and, upon annealing of an anti-sense promoter oligo, is used as a linear transcription subsfrate for synthesis of RNA using the RNA polymerase that initiates transcription from said promoter under transcription conditions. Synthesis of RNA corresponding to the target sequence and/or the signal sequence in these linear transcription subsfrates can be detected according to the detection method used in the particular embodiment of an assay or method ofthe invention.
  • the first sfrand cDNA can be used as a ligation template for ligation of a second bipartite target probe under ligation conditions.
  • the second bipartite target probe is identical to the first bipartite target probe except that with respect to the target- complementary sequences at the 3'- and 5'-ends of said second bipartite target probe.
  • the 5'- end portion ofthe second bipartite target probe comprises a sequence that is complementary to the target-complementary sequence at the 3 '-end portion ofthe first bipartite target probe, and this sequence is in turn covalently attached and 5 '-of a promoter sequence in the 5 '-portion of the second bipartite target probe.
  • the 3 '-end portion ofthe second bipartite target probe comprises a sequence that is complementary to the target-complementary sequence at the 5 '-end portion ofthe first bipartite target probe, and this sequence is in turn covalently attached and 3'- of a signal sequence in the 3 '-portion ofthe second bipartite target probe, if a signal sequence is present.
  • sequences at the 3'- and 5 '-ends of said second bipartite target probe are identical to the target sequence and are complementary to the target-complementary sequences in both the first circular transcription substrate and in the first-strand cDNA obtained by reverse transcription of RNA transcripts from said first circular franscription substrate, both of which thus serve as ligation templates for ligation ofthe second bipartite target probe by a ligase under ligation conditions.
  • Ligation of a second bipartite target probe and annealing of an anti-sense promoter oligo generates a second circular transcription substrate.
  • the second circular transcription subsfrate is then a subsfrate for rolling circle transcription, generating a complementary RNA multimer transcript.
  • the RNA multimer transcript resulting from rolling circle franscription is then a substrate for reverse franscription by a reverse franscriptase under reverse franscription conditions. Since, in the embodiment shown in Figure 9, the second circular franscription subsfrate is identical to the first circular franscription substrate in all portions except for the target-complementary portion, the same reverse franscription primer can be used to generate first-sfrand cDNA that is complementary to the RNA multimer from the second circular transcription subsfrate.
  • the resulting first-sfrand cDNA after annealing of an anti-sense promoter oligo, is a second linear franscription subsfrate.
  • In vitro franscription of said second linear franscription subsfrate by an RNA polymerase that initiates franscription using said double-sfranded transcription promoter under transcription conditions generates RNA transcripts that can be detected in the assay or method.
  • the sequence corresponding to a target sequence in said first-sfrand cDNA also serves as a template for ligation of a first bipartite target probe by a ligase under ligation conditions.
  • one embodiment ofthe present invention comprises a method for detecting a target sequence, said method comprising: [00363] a. providing a first bipartite target probe comprising linear single-sfranded DNA having two target-complementary sequences that are not joined to each other and that are contiguous when annealed to the target sequence, wherein the 5 '-end ofthe first target- complementary sequence is complementary to the 5 '-end ofthe target nucleic acid sequence, and wherein the 3 '-end of a second target-complementary sequence is complementary to the 3'- end ofthe target nucleic acid sequence, and wherein the 3'-end ofthe first target-complementary sequence is joined to the 5 '-end of a sense promoter sequence for an RNA polymerase; [00364] b. providing a second bipartite target probe comprising linear single-sfranded
  • RNA that is complementary to said first circular transcription substrate a primer, wherein said primer is complementary to said RNA
  • n annealing to said RNA that is complementary to said second circular transcription subsfrate a primer, wherein said primer is complementary to said RNA;
  • RNA RNA resulting from franscription of said first, second and third circular transcription substrates and from said first and second linear franscription substrates, wherein said synthesis of said RNA indicates the presence of said target sequence.
  • the circular transcription substrates that are transcribed remain catenated during franscription.
  • one or more additional steps are used in order to release the catenated circular ligation products from the target sequence when the target probe anneals to a sequence in a linear DNA molecule that is greater than about 150 to about 200 nucleotides from the 3'-end ofthe linear DNA molecule, as discussed elsewhere herein.
  • DNA polymerization or reverse transcription is carried out using a ratio of dUTP to dTTP that results in inco ⁇ oration of one dUMP residue about every 200-400 nucleotides and a composition comprising uracil-N-glycosylase and endonuclease IV is used to release catenated DNA molecules following ligation of bipartite target annealed to the long linear DNA molecules.
  • the ligase has little or no activity in ligating blunt ends and is substantially more active in ligating ends that are adjacent when annealed to a contiguous complementary sequence compared to ends that are not adjacently annealed to acomplementary sequence.
  • One suitable ligase that can be used is Ampligase® Thermostable DNA Ligase (EPICENTRE Technologies, Madison, WI).
  • One suitable reverse franscriptase that can be used is MMLV Reverse Transcriptase.
  • Another suitable reverse transcriptase that can be used is IsoThermTM DNA Polymerase (EPICENTRE Technologies, Madison, WI).
  • RNA polymerases are T7 RNAP, T3 RNAP, SP6 RNAP or another T7-like RNA polymerase or a mutant form of one of these T7-like RNA polymerases.
  • AmpliScribe T7-FlashTM Transcription Kit is used for in vitro franscription ofthe transcription substrate (EPICENTRE Technologies, Madison, WI).
  • the anti-sense promoter oligo is attached to a solid support.
  • the anti-sense promoter oligo comprises a moiety, such as, but not limited to a biotin moiety that permits binding ofthe anti-sense promoter oligo to a solid support after annealing to the ligation product to obtain a transcription subsfrate.
  • the target sequence comprises a target nucleic acid in a sample, whereas in other embodiments the target sequence comprises a target sequence tag that is joined to an analyte- binding substance that binds an analyte in the sample.
  • one embodiment ofthe present invention comprises a method for detecting a target sequence, said method comprising: [00390] 1. providing a reaction mixture comprising:
  • a first bipartite target probe wherein said first bipartite target probe comprises a 5'-portion and a 3'-portion, wherein said 5'-portion comprises: (i) a 5'-end portion that comprises a 5 '-phosphate group and a sequence that is complementary to a target sequence, and (ii) a sense promoter sequence, wherein said sense promoter sequence is covalently attached to and 3'-of said target-complementary sequence in said 5'-portion; and wherein said 3'-portion comprises: (i) a 3'-end portion that comprises a sequence that is complementary to a target sequence, wherein said target-complementary sequence of said 3 '-end portion, when annealed to said target sequence, is adjacent to said target-complementary sequence of said 5 '-end portion of said first bipartite target probe, and (ii) optionally, a signal sequence, wherein said signal sequence is 5'-of said target-complementary sequence of said 3'
  • a second bipartite target probe wherein said second bipartite target probe comprises a 5 '-portion and a 3 '-portion, wherein said 5 '-portion comprises: (i) a 5 '-end portion that comprises a 5 '-phosphate group and sequence that is complementary to said target- complementary sequence of said 3 '-end portion of said first bipartite target probe, and (ii) a sense promoter sequence, wherein said sense promoter sequence in said 5 '-portion of said second bipartite target probe is 3 '-of said target-complementary sequence in said 5 '-portion; and wherein said 3 '-portion comprises: (i) a 3 '-end portion that comprises sequence that is complementary to said target-complementary sequence of said 5 '-end portion of said first bipartite target probe, and (ii) optionally, a signal sequence, wherein said signal sequence in said 3'-portion of said second bipartite target probe is 5'-of said
  • a reverse transcriptase and one or more primers wherein at least the 3 '-portion of one said primer comprises a sequence that is complementary to a sequence of said first bipartite target probe and of said second bipartite target probe and wherein said complementary portion of said primer is not complementary to said target sequence or the complement of said target sequence;
  • RNA polymerase recognizes said single- sfranded transcription promoters of said first and second bipartite target probes and synthesizes RNA therefrom using as a template single-sfranded DNA to which said promoters are functionally attached;
  • a detection oligo wherein said detection oligo anneals to an RNA transcript sequence that is complementary to a signal sequence of said first and/or second bipartite target probe;
  • RNA polymerase are optimally active in combination and wherein said target-complementary sequences of said first bipartite target probe anneal to said target sequence, if present, with specificity, and;
  • said ligase comprises a ligase chosen from among Ampligase® thermostable DNA Ligase, Tth DNA Ligase, Tfl DNA Ligase, Tsc DNA Ligase, or Pfu DNA Ligase.
  • the compositions that result in release of catenated DNA molecules that are ligated on a linear template comprise nucleotides comprising a ratio of dUTP to dTTP that results in inco ⁇ oration of a dUMP residue about every 200-400 nucleotides and a composition comprising uracil-N-glycosylase and endonuclease TV.
  • said reverse franscriptase is a reverse transcriptase that has RNase H activity, wherein said reverse franscriptase is chosen from among MMLV reverse franscriptase, AMV reverse transcriptase, another retroviral reverse transcriptase, or a reverse transcriptase encoded by a thermostable phage.
  • said reverse transcriptase comprises a DNA polymerase chosen from among IsoThermTM DNA polymerase, Bst DNA polymerase large fragment, Bca.BESTTM DNA polymerase, and Tth DNA polymerase.
  • RNA polymerase is T7 RNAP, T3 RNAP or SP6 RNAP.
  • the methods using target probes that comprise a sense promoter sequence for a double-sfranded promoter described in this section above can also be used to detect a target sequence tag that is joined to an analtye-binding substance, examples of which are illustrated in Figures 10 and 11 , and which are described in greater detail in the next section pertaining to detection of non-nucleic acid analytes.
  • the present invention also comprises embodiments that use an RNA polymerase that recognizes a cognate single-sfranded franscription promoter or a single-sfranded pseudopromoter.
  • the promoter sequence in a monopartite promoter target probe or in a bipartite target probe comprises a sequence for the single-stranded promoter or pseudopromoter recognized by the cognate RNA polymerase.
  • a prefened single-stranded promoter comprises an N4 promoter and the cognate RNA polymerase that is used is an N4 mini-vRNAP or a Y678F mutant of an N4 mini-vRNAP.
  • Prefened single-stranded pseudopromoters include a pseudopromoter or synthetic single- sfranded promoter for a T7-type RNA polymerase, chosen from among T7 RNAP, T3 RNAP or SP6 RNAP, or for E. coli RNAP or for ⁇ iermus thermophilus RNAP, and the cognate RNA polymerase for the promoter is used.
  • Some pseudopromoters that can be used in a promoter target probe or a bipartite target probe if E. coli RNAP is used are provided in Ohmichi et al. (Proc. Natl. Acad. Sci. USA, 99: 54-59, 2002).
  • the present invention includes methods, compositions and kits that use an analyte-binding substance for detecting an analyte in a sample.
  • An "analyte-binding substance” is a substance that binds an analyte that one desires to detect in an assay or method ofthe invention.
  • An analyte-binding substance is also referred to as an "affinity molecule,” an “affinity substance,” a “specific binding substance,” or a "binding molecule” for an analyte.
  • an analyte molecule and an analyte-binding substance or affinity molecule for the analyte molecule are related as a specific "binding pair", i.e., their interaction is only through non-covalent bonds such as hydrogen-bonding, hydrophobic interactions (including stacking of aromatic molecules), van der Waals forces, and salt bridges. Without being bound by theory, it is believed in the art that these kinds of non-covalent bonds result in binding, in part due to complementary shapes or structures ofthe molecules involved in the binding pair.
  • binding refers to the interaction between an analyte-binding substance or affinity molecule and an analyte as a result of non-covalent bonds, such as, but not limited to, hydrogen bonds, hydrophobic interactions, van der Waals bonds, and ionic bonds.
  • target probes are used to detect an analyte comprising a target sequence in a target nucleic acid. Following annealing and joining of target probes in the presence of a target sequence in a sample, the resulting transcription subsfrate is amplified by franscription using an RNA polymerase, and the presence of an RNA complementary to the transcription substrate indicates that the target sequence was present in the sample.
  • a nucleic acid can also be used in a method ofthe present invention as an analyte-binding substance to detect an analyte that does not comprise a nucleic acid.
  • a method termed "SELEX,” as described by Gold and Tuerk in U.S. Patent No. 5,270,163, which is inco ⁇ orated herein by reference can be used to select a nucleic acid for use as an analyte-binding substance in a method ofthe invention for detecting an analyte comprising almost any molecule in a sample.
  • SELEX permits selection of a nucleic acid molecule that has high affinity for a specific analyte from a large population of nucleic acid molecules, at least a portion of which have a randomized sequence. For example, a population of all possible randomized 25-mer oligonucleotides (i.e., having each of four possible nucleic acid bases at every position) will contain 4 25 (or 10 15 ) different nucleic acid molecules, each of which has a different three-dimensional structure and different analyte binding properties. SELEX can be used, according to the methods described in U.S. Patent Nos.
  • analyte-binding substances or affinity molecules comprising nucleic acid molecules can be made for use in the methods ofthe present invention by using any of numerous in vivo or in vitro techniques known in the art, including, by way of example, but not of limitation, automated nucleic acid synthesis techniques, PCR, or in vitro franscription.
  • a nucleic acid molecule that is an analyte-binding substance that has been selected using SELEX can be defected using bipartite or monopartite target probes in a similar way to how such target probes are used to detect a target sequence in a target nucleic acid analyte, as described elsewhere herein.
  • an analyte-binding substance that is selected using SELEX comprises a nucleic acid
  • a continuous sequence within the analyte-binding substance can be used as a "target sequence” and target probes can be designed, wherein the target-complementary sequences in said target probes are complementary to said continuous sequence in said analyte- binding substance.
  • target sequence in said analyte-binding substance that was selected using SELEX should be capable of annealing to said target probes when said analyte-binding substance is also bound to an analyte; i.e., the binding to the analyte does not block annealing of target probes to the target sequence.
  • another embodiment ofthe present invention is a method for detecting an analyte in a sample, ' wherein said analyte comprises a biomolecule that is not a nucleic acid, said method comprising:
  • an analyte-binding substance comprising a nucleic acid, wherein said nucleic acid binds with selectivity and high affinity to said analyte;
  • target probes comprising either (i) a promoter target probe and one or more additional target probes chosen from among a signal target probe and simple target probe; or (ii) a bipartite target probe and, if said target-complementary sequences of said bipartite target probe are not contiguous when annealed to said target sequence in said analyte- binding substance, optionally, one or more simple target probes; wherein said target probes of (i) or (ii) comprise sequences that are complementary to adjacent regions of a target sequence in said analyte-binding substance;
  • an analyte-binding substance comprising a nucleic acid selected using SELEX permits the methods ofthe present invention to be used to detect other analyte molecules that are not nucleic acids.
  • nucleic acid molecules that contain a randomized sequence that are used to generate a library of molecules for selection of an analyte-binding substance using SELEX can also be made using methods similar to those described by Ohmichi et al. (Proc. Natl. Acad. Sci. USA, 99: 54-59, 2002), inco ⁇ orated herein by reference.
  • random sequence circular DNA molecules comprising about 103 nucleotides, of which about 40 nucleotides comprise randomized sequence are repeatedly selected for binding to an analyte by: binding the circular DNAs to an analyte attached to a surface; washing away the unbound circular DNA molecules; recovering the circular DNAs bound to the analyte; obtaining RNA complementary to the recovered circular DNA molecules by rolling circle transcription; amplifying the RNA by RT- PCR using one 5'-biotinylated primer; immobilizing the RT-PCR product on a surface with streptavidin; obtaining the strand ofthe RT-PCR product that does not contain biotin; and then ligating the single-sfranded RT-PCR strand (using a ligation splint) to obtain the first round of selected circular DNA molecules.
  • the first round of circular DNA molecules is then bound to an analyte as just described, and the whole process is again repeated for a total of about 15 rounds of selection of circular DNA molecules for analyte binding.
  • the selected circular DNA molecules are then analyzed for analyte binding in order to obtain an analyte-binding substance for use in an assay or method o the present invention.
  • a target sequence in the selected analyte-binding substance can be detected using monopartite or bipartite target probes as described elsewhere herein.
  • an analyte-binding substance is used to bind an analyte in a sample and then, after removing unbound analyte-binding substance (if the analyte-binding substance is attached to a surface or becomes attached to a surface during a process ofthe assay or method), the analyte-binding substance is detected using target probes that are complementary to a target sequence in the analyte-binding substance.
  • a method for detecting an analyte-binding substance can comprise a step comprising ligation of target probes ofthe invention as described in the embodiments ofthe method immediately above herein for detecting a target sequence in an analyte-binding substance that is bound to an analyte.
  • a ligation step is omitted and the analyte-binding substance:analyte complex is detected by annealing to said complex a franscription substrate that contains a sequence that is complementary to a target sequence in said analyte-binding substance.
  • transcription substrates that are annealed to said analyte- binding substance: analyte complex are detected by synthesis of RNA resulting from in vitro transcription ofthe complex-bound transcription substrate.
  • a PNA molecule is a nucleic acid analog consisting of a backbone comprising, for example, N-(2-aminoethyl)glycine units, to each of which a nucleic acid base is linked through a suitable linker, such as, but not limited to an aza, amido, ureido, or methylene carbonyl linker.
  • the nucleic acid bases in PNA molecules bind complementary single-sfranded DNA or RNA according to Watson-Crick base-pairing rules.
  • the T m 's for PNA/DNA or PNA/RNA duplexes or hybrids are higher than the T m 's for DNA DNA, DNA/RNA, or RNA RNA duplexes.
  • a "PNA target sequence” is present in said analyte- binding substance comprising PNA, to which, target-complementary sequences of monopartite or bipartite target probes (or transcription subsfrates) can anneal, permitting detection as described above for analyte-binding molecules selected using SELEX.
  • PNA used as an analyte-binding substance in an assay or method ofthe present invention provides tighter binding (and greater binding stability) for target-complementary sequences in target probes or franscription substrates (e.g., see U.S. Patent No. 5,985,563).
  • PNA molecules are highly resistant to protease and nuclease activity.
  • PNA for use as an analyte binding substance can be prepared according to methods known in the art, such as, but not limited to, methods described in the above-mentioned patents, and references therein.
  • Antibodies to PNA/analyte complexes can be used in the invention for capture, recognition, detection, identification, or quantitation of nucleic acids in biological samples, via their ability to bind specifically to the respective complexes without binding the individual molecules (U.S. Patent No. 5,612,458).
  • the invention also contemplates that a combinatorial library of randomized peptide nucleic acids prepared by a method such as, but not limited to, the methods described in U.S. Patent Nos. 5,539,083; 5,831,014; and 5,864,010, can be used to prepare analyte-binding substances for use in assays for analytes of all types, including analytes that are nucleic acids, proteins, or other analytes, without limit.
  • randomized peptide or peptide nucleic acid libraries are made to contain molecules with a very large number of different binding affinities for an analyte.
  • an analyte-binding substance can also be an oligonucleotide or polynucleotide with a modified backbone that is not an amino acid, such as, but not limited to modified oligonucleotides described in U.S. Patent Nos. 5,602,240; 6,610,289; 5,696,253; or 6,013,785.
  • an analyte-binding substance can be prepared from a combinatorial library of randomized peptides (i.e., comprising at least four naturally-occurring amino acids).
  • One way to prepare the randomized peptide library is to place a randomized DNA sequence, prepared as for SELEX, downstream of a phage T7 RNA polymerase promoter, or a similar promoter, and then use a method such as, but not limited to, coupled transcription-translation, as described in U.S. Patent Nos. 5,324,637; 5,492,817; or 5,665,563, or stepwise transcription, followed by translation.
  • a randomized DNA sequence prepared as for SELEX, can be cloned into a site in a DNA vector that, once inserted, encodes a recombinant MDV-1 RNA containing the randomized sequence that is replicatable by Q-beta replicase (e.g., between nucleotides 63 and 64 in MDV-1 (+) RNA; see U.S. Patent No. 5,620,870).
  • the recombinant MDV-1 DNA containing the randomized DNA sequence is downstream from a T7 RNA polymerase promoter or a similar promoter in the DNA vector.
  • the recombinant MDV-1 RNA, containing the randomized sequence can be used to make a randomized peptide library comprising at least four naturally occurring amino acids by coupled replication-translation as described in U.S. Patent No. 5,556,769.
  • An analyte-binding substance can be selected from the library by binding peptides in the library to an analyte, separating the unbound peptides, and identifying one or more peptides that is bound to analyte by means known in the art.
  • high throughput screening methods can be used to screen all individual peptides in the library to identify those that can be used as analyte-binding substances.
  • an analyte-binding substance comprises a peptide, a protein, including, but not limited to an antibody, sfreptavidin, or another biomolecule
  • a nucleic acid sequence can be attached to said analyte-binding substance, wherein said nucleic acid serves as a "tag" comprising a target sequence that can be detected using target probes or transcription substrates ofthe invention.
  • the methods and assays ofthe invention can be used for sensitive and specific detection of analytes that are not nucleic acids.
  • Analyte-binding substances for particular analytes and methods of preparing them are well known in the art.
  • nucleic acid sequences such as operators, promoters, origins of replication, sequences recognized by steroid hormone-receptor complexes, restriction endonuclease recognition sequences, ribosomal nucleic acids, and so on, which are known to bind tightly to certain proteins.
  • the lac repressor and the bacteriophage lambda repressor each bind to their respective specific nucleic acid sequences called "operators" to block initiation of transcription of their corresponding mRNA molecules.
  • Nucleic acids containing such specific sequences can be used in the invention as analyte-binding substances for the respective proteins or other molecules for which the nucleic acid has affinity.
  • the nucleic acid with the specific sequence is used as the analyte-binding substance in assays for the respective specific protein, glycoprotein, lipoprotein, small molecule or other analyte that it binds.
  • One of several techniques that is generally called “footprinting" e.g., see Galas, D.
  • an analyte-binding substance can be an antibody, including monoclonal, polyclonal, or artificial antibodies which are made using methods well known in the art
  • tha analyte can be any substance for which a specific-binding antibody can be prepared, including peptides, proteins, carbohydrates, lipids glycoproteins, lipoproteins, and biochemicals, either alone or conjugated to another molecule in order to increase the "antigenicity," or ability to provoke an antibody response.
  • antibodies, including monoclonal antibodies are available as analyte- binding substances.
  • an antibody binding protein such as Staphylococcus aureus Protein A can be employed as an analyte-binding substance.
  • an analyte such as a glycoprotein or class of glycoproteins, or a polysaccharide or class of polysaccharides, which is distinguished from other substances in a sample by having a carbohydrate moiety that is bound specifically by a lectin
  • a suitable analyte- binding substance is the lectin.
  • a receptor for the hormone can be employed as an analyte-binding substance.
  • an analyte that is a receptor for a hormone can be employed as the analyte-binding substance.
  • an inhibitor ofthe enzyme can be employed as an analyte-binding substance.
  • the enzyme can be employed as the analyte-binding substance.
  • binding conditions vary for different specific binding pairs. Those skilled in the art can easily determine conditions whereby, in a sample, binding occurs between affinity molecule and analyte that may be present. In particular, those skilled in the art can easily determine conditions whereby binding between affinity molecule and analyte that would be considered in the art to be "specific binding” can be made to occur. As understood in the art, such specificity is usually due to the higher affinity of affinity molecule for analyte than for other substances and components (e.g., vessel walls, solid supports) in a sample. In certain cases, the specificity might also involve, or might be due to, a significantly more rapid association of affinity molecule with analyte than with other substances and components in a sample.
  • any ofthe methods and assays described herein to detect and quantify an analyte comprising a target sequence in a target nucleic acid can also be used to detect and quantify a target sequence that comprises a target sequence tag that is attached to an analyte- binding substance for a non-nucleic acid analyte by adjusting the reaction conditions of said assay or method to accommodate the specific analyte and analyte-binding substance.
  • the methods and assays ofthe invention permit detection and quantification of any analyte for which there is a suitable analyte-binding substance that either comprises or to which a target sequence tag can be attached.
  • the invention also comprises methods, compositions and kits for using ssDNA transcription subsfrates and RNA polymerases that can transcribe said ssDNA franscription substrates as a signaling system for an analyte of any type, including analytes such as, but not limited to, antigens, antibodies or other substances, in addition to an analyte that is a target nucleic acid.
  • analytes such as, but not limited to, antigens, antibodies or other substances, in addition to an analyte that is a target nucleic acid.
  • the invention comprises a method for detecting an analyte in or from a sample, said method comprising:
  • a transcription signaling system comprising a ssDNA comprising: (a) a 5 '-portion comprising a sense promoter sequence for a double-sfranded promoter for a cognate RNA polymerase; and (b) a signal sequence, wherein said signal sequence, when transcribed by said RNA polymerase, is detectable in some manner; [00439] 2.
  • RNA polymerase synthesizes RNA that is complementary to said signal sequence in said ssDNA transcription signaling system under said franscription conditions
  • An analyte or an analyte-binding substance of this aspect ofthe invention can be any combination of biological molecules that can form a specific binding pair.
  • an analyte-binding substance can be an antibody and the analyte an antigen, or an analyte-binding substance can be a nucleic acid and the analyte can be another complementary nucleic acid.
  • the signal sequence can vary greatly.
  • a signal sequence can comprise a substrate for Q-beta replicase, which is detectable in the presence of said replicase under replication conditions.
  • It can also comprise a sequence that encodes a protein, such as green fluorescent protein, that is detectable following translation ofthe signal sequence.
  • a probe such as, but not limited to a molecular beacon, as described by Tyagi et al. (U.S. Patents Nos. 5,925,517 and 6,103,476 of Tyagi et al. and 6,461,817 of Alland et al., all of which are inco ⁇ orated herein by reference).
  • the present invention with regard to signaling systems also comprises uses such as those for methods described by Zhang et al. (Proc. Natl. Acad. Sci. USA, 98: 5497-5502, 2001, inco ⁇ orated herein by reference) or by Hudson et al. in U.S. Patent No. 6,100,024, inco ⁇ orated herein by reference.
  • the methods ofthe invention can be performed in a stepwise fashion, with one set of reactions being performed, followed by purification of a reaction product or removal of reagents or inactivation of enzymes or addition of reagents before proceeding to the next set of reactions, or, in other embodiments, which are prefened embodiments, the methods and assays can be performed as a continuous set of multiple reactions in a single reaction mixture.
  • the invention also comprises methods or assays in which multiple target probes or target probe sets are used in a single reaction mixture in order to detect and/or quantify multiple target sequences in one or multiple target nucleic acids.
  • the compositions, kits, methods and assays ofthe invention can be used in a multiplex format.
  • the invention is not limited to these reaction conditions or concenfrations of reactants, except that the reaction conditions must be appropriate for each step of a method or assay ofthe invention.
  • Those with skill in the art will know how to find and determine suitable reaction conditions under which enzymes, including ligases, RNA polymerases, DNA polymerases, replicases (such as Q-beta replicase) and related enzymes are active for the methods ofthe invention and will know that optimal combined conditions can be used can be found by simple experimentation, and any of these reaction conditions are included within the scope ofthe invention.
  • Such media and conditions are known to persons of skill in the art, and are described in various publications such as, but not limited to U.S. Pat. No. 5,679,512 and PCT Pub.
  • a buffer can be Tris buffer, although other buffers can also be used as long as the buffer components are non-inhibitory to enzyme components ofthe methods ofthe invention.
  • the pH is preferably from about 5 to about 11, more preferably from about 6 to about 10, even more preferably from about 7 to about 9, and most preferably from about 7.5 to about 8.5.
  • the reaction medium can also include bivalent metal ions such as Mg +2 or Mn , at a final concentration of free ions that is within the range of from about 0.01 to about 10 mM, and most preferably from about 1 to 6 mM.
  • the reaction medium can also include other salts, such as KC1, that contribute to the total ionic sfrength ofthe medium.
  • the range of a salt such as KC1 is preferably from about 0 to about 100 mM, more preferably from about 0 to about 75 mM, and most preferably from about 0 to about 50 mM.
  • Cofactors can be supplied for enzymes as appropriate, such as, but not limited to NAD at a final concentration of about 0.5 mM for an NAD-dependent ligase or ATP at a final concentration of about 0.1 to 1.0 mM for an ATP-dependent ligase or a polynucleotide kinase, respectively.
  • the reaction medium can further include additives that could affect performance ofthe reactions, but that are not integral to the activity ofthe enzyme components ofthe methods.
  • Such additives include proteins such as BS A, and non-ionic detergents such as NP40 or Triton.
  • Reagents such as DTT, that are capable of maintaining activities enzyme with sulfhydryl groups can also be included.
  • DTT agent capable of maintaining activities enzyme with sulfhydryl groups
  • Such reagents are known in the art.
  • an RNase inhibitor such as, but not limited to a placental ribonuclease inhibitor (e.g., RNasin®, Promega Co ⁇ oration, Madison, WI, USA) or an antibody RNase inhibitor, that does not inhibit the activity of an RNase employed in the method can also be included. Any aspect ofthe methods ofthe present invention can occur at the same or varying temperatures.
  • the reactions are performed isothermally, which avoids the cumbersome thermocycling process.
  • the reactions are carried out at a temperature that permits hybridization ofthe oligonucleotides ofthe present invention to the target sequence and/or first-sfrand cDNA of a method ofthe invention and that does not substantially inhibit the activity ofthe enzymes employed.
  • the temperature can be in the range of preferably about 25° C to about 85° C, more preferably about 30° C to about 75° C, and most preferably about 37° C to about 70° C.
  • the temperature for the transcription steps is lower than the temperature(s) for the preceding steps.
  • the temperature ofthe transcription steps can be in the range of preferably about 25° C to about 85° C, more preferably about 30° C to about 75° C, and most preferably about 37° C to about 55° C.
  • Patent No. DE4411588C1 all of which are inco ⁇ orated herein by reference and made part of the present invention, it is preferred in many embodiments to use a final concentration of about 0.25 M, about 0.5 M, about 1.0 M, about 1.5 M, about 2.0 M, about 2.5 M or between about 0.25 M and 2.5 M betaine (trimethylglycine) in DNA polymerase or reverse transcriptase reactions in order to decrease DNA polymerase stops and increase the specificity of reactions which use a DNA polymerase.
  • betaine trimethylglycine
  • Nucleotide and/or nucleotide analogs such as deoxyribonucleoside triphosphates, that can be employed for synthesis of reverse franscription or primer extension products in the methods ofthe invention are provided in an amount that is determined to be optimal or useful for a particular intended use.
  • the oligonucleotide components of reactions ofthe invention are generally in excess ofthe number of target nucleic acid sequence to be amplified. They can be provided at about or at least about any ofthe following: 10, 10 2 , 10 4 , 10 6 , 10 8 , 10 10 , 10 12 times the amount of target nucleic acid.
  • Target probes, primers, anti-sense promoter oligos, strand-displacement primers, and the like can each be provided at about or at least about any ofthe following concentrations: 50 nM, 100 nM, 500 nM, 1000 nM, 2500 nM, 5000 nM, or 10,000 nM, but higher or lower concenfrations can also be used.
  • a concentration of one or more oligonucleotides may be desirable for production of one or more target nucleic acid sequences that are used in another application or process.
  • the invention is not limited to a particular concentration of an oligonucleotide, so long as the concentration is effective in a particular method ofthe invention.
  • the foregoing components are added simultaneously at the initiation ofthe process.
  • components are added in any order prior to or after appropriate time points during the process, as required and/or permitted by the reaction. Such time points can readily be identified by a person of skill in the art.
  • the enzymes used for nucleic acid reactions according to the methods ofthe present invention are generally added to the reaction mixture following a step for denaturation of a double-sfranded target nucleic acid in or from a sample, and/or following hybridization of primers and/or oligos of a reaction to a denatured double-sfranded or single-sfranded target nucleic acid, as determined by their thermal stability and/or other considerations known to the person of skill in the art.
  • the reactions can be stopped at various time points, and resumed at a later time.
  • the time points can readily be identified by a person of skill in the art.
  • Methods for stopping the reactions are known in the art, including, for example, cooling the reaction mixture to a temperature that inhibits enzyme activity.
  • Methods for resuming the reactions are also known in the art, including, for example, raising the temperature ofthe reaction mixture to a temperature that permits enzyme activity.
  • one or more ofthe components ofthe reactions is replenished prior to, at, or following the resumption ofthe reactions.
  • the reaction can be allowed to proceed (i.e., from start to finish) without interruption.
  • the invention also comprises parts or subsets ofthe methods and compositions of the invention.
  • the invention comprises all ofthe individual steps ofthe methods ofthe invention that are enabled thereby, in addition to the overall methods.
  • kits and compositions for carrying out the methods ofthe invention also comprises kits and compositions for carrying out the methods ofthe invention.
  • a kit ofthe invention comprises one or, preferably, multiple components or compositions for carrying out the various processes of a method.
  • Different embodiments of kits and compositions ofthe present invention can comprise one or more ofthe following:
  • a bipartite target probe for an assay or method for detecting a particular target sequence, and, optionally if the target-complementary sequences of said bipartite target probe are not contiguous when annealed to a target sequence, a monopartite target probe, all of which target probes preferably have a 5 '-phosphate group.
  • a set of monopartite target probes for an assay or method for detecting a particular target sequence wherein said set of monopartite target probes comprises a promoter target probe, preferably having a 5 '-phosphate group, and either a signal target probe or a simple target probe and one or more additional simple target probes, which if present, preferably each have a 5 '-phosphate group.
  • a ligase wherein said ligase has little or no activity in ligating blunt ends and is substantially more active in ligating the ends of target probe if said ends are adjacent when annealed to two contiguous regions of a complementary sequence than if said ends are not annealed to said complementary sequence.
  • said ligase comprises a ligase chosen from among Ampligase® thermostable DNA Ligase, Tth DNA Ligase, Tfl DNA Ligase, Tsc DNA Ligase, or Pfu DNA Ligase.
  • RNA polymerase preparation wherein said RNA polymerase recognizes a transcription promoter in a franscription substrate generated from a method ofthe invention and initiates transcription therefrom using as a template single-sfranded DNA to which said promoter is functionally attached.
  • said RNA polymerase is T7 RNAP, T3 RNAP or SP6 RNAP.
  • E. coli RNAP or Thermus thermophilus RNAP is used.
  • a mutant enzyme such as, but not limited to T7 Y639F mutant RNAP is preferred.
  • the kit or composition also comprises an anti-sense promoter oligo, which in some embodiments is attached to a solid support.
  • the RNA polymerase preparation comprises an N4 mini-vRNAP and EcoSSB Protein.
  • said reverse transcriptase has RNase H activity and is chosen from among MMLV, AMV or another refroviral reverse franscriptase, or a reverse franscriptase encoded by a thermostable phage.
  • said reverse transcriptase comprises a DNA polymerase chosen from among IsoThermTM, Bst large fragment, Bca.B ⁇ STTM, and Tth DNA polymerase.
  • primers which can also comprise a composition or kit ofthe invention.
  • any DNA polymerase that does not strand displace a downstream target probe can be used in a composition or kit ofthe invention.
  • an analyte-binding substance that either comprises or has an attached target sequence tag for embodiments ofthe invention for detecting and/or quantifying an analyte in a sample.
  • a composition or kit ofthe invention can comprise a wide variety of compositions for detecting an RNA transcript that is complementary to a target sequence and/or a signal sequence.
  • a composition or kit can comprise a detection oligo, such as a molecular beacon.
  • a composition or kit can comprise an enzyme, such as Q-beta replicase, if the signal sequence encodes a Q-beta replicase subsfrate.
  • the invention comprises kits comprising any suitable detection composition.
  • Controls are used in assays and methods ofthe invention in order to verify that the assay or method produces the required specificity and sensitivity, or, in other words, to determine the frequency and conditions that lead to "false positive” and/or "false negative” results.
  • controls comprise important compositions and kits for assays and methods ofthe invention.
  • a positive control might be a sample containing a known quantity of a target sequence.
  • a negative control would lack the target sequence.
  • positive controls might comprise sample that contain either the mutant or the predominant allele or other known alleles for that nucleotide position in the target sequence.
  • quantification of a target analyte in a sample using an assay or method ofthe invention, including an analyte comprising a target sequence is achieved by using controls containing different known quantities of said analyte as a standard.
  • the control sample is as close in performance as possible to a "real world" sample using the methods or assays ofthe invention, the amount ofthe analyte in the sample can be standardized against the results obtained using quantification controls.
  • a composition or kit can also, for example, comprise a confrol comprising an antigen for an assay or method that uses an analyte-binding substance comprising an antibody with a bound target sequence tag or a molecule selected using SELEX.
  • Most ofthe embodiments for detecting a target sequence are linear and the side-by-side results obtained compared to quantification standards will be proportional to the amount of said analyte in a sample.
  • special care will need to be taken in trying to quantify the amount of analyte in a sample when an embodiment of an assay or method that comprises secondary or additional amplification processes, such as, the embodiment illustrated in Figure 9.
  • kits ofthe invention will also comprise a description ofthe components of said kit and instructions for their use in a particular process or method or methods ofthe invention.
  • a kit ofthe present invention will also comprise other components, such as, but not limited to, buffers,.ribonucleotides and/or deoxynucleotides, including modified nucleotides in some embodiments, DNA polymerization or reverse franscriptase enhancers, such as, but not limited to betaine (trimethylglycine), and salts of monvalent or divalent cations, such as but not limited to potassium acetate or chloride and/or magnesium chloride, enzyme substrates and/or cofactors, such as, but not limited to, ATP or NAD, and the like which are needed for optimal conditions of one or more reactions or processes of a method or a combination of methods for a particular application.
  • buffers such as, but not limited to, buffers,.ribonucleotides and/or deoxynucleotides, including modified nucleot
  • a kit ofthe invention can comprise a a set of individual reagents for a particular process or a series of sets of individual reagents for multiple processes of a method that are performed in a stepwise or serial manner, or a kit can comprise a multiple reagents combined into a single reaction mixture or a small number of mixtures of multiple reagents, each of which perform multiple reactions and or processes in a single tube.
  • the various components of a kit for performing a particular process of a method ofthe invention or a complete method ofthe invention will be optimized so that they have appropriate amounts of reagents and conditions to work together in the process and/or method.
  • a "ligation splint" or a “ligation splint oligo” is an oligo that is used to provide an annealing site or a "ligation template" for joining two ends of one nucleic acid (i.e., "intramolecular joining") or two ends of two nucleic acids (i.e., "intermolecular joining”) using a ligase or another enzyme with ligase activity.
  • the ligation splint holds the ends adjacent to each other and “creates a ligation junction" between the 5 '-phosphorylated and a 3'-hydroxylated ends that are to be ligated.
  • the invention also comprises embodiments of target-dependent franscription in which a circular franscription substrate comprising a target-complementary sequence is generated even if there is no target-complementary sequence at either the 3'-end or the 5'-end or at both ends of an "open circle probe" (the word "target” is removed from the name ofthe probe here because there are no target-complementary sequences).
  • An open circle probe ofthe invention comprises an oligonucleotide having a 5 '-end portion comprising a sequence for a sense promoter sequence for a cognate RNA polymerase that uses a double-sfranded promoter or, in other embodiments, a single-sfranded promoter or pseudopromoter for a cognate RNA polymerase and a 3'-end portion comprising a sequence that is not a promoter sequence, which sequene can optionally comprise a signal sequence, as discussed elsewhere herein. [00468] In embodiments that use an open circle probe, simple target probes that can anneal to the target sequence are used.
  • the resulting linear ligation product comprising a target-complementary sequence is joined directly to an open circle probe using two ligation splints, each of which has a portion complementary to a respective end ofthe open circle probe and to an appropriate end ofthe target-complementary sequence in the linear ligation product.
  • One ligation splint oligo is used to join a sense promoter of an open circle probe to the 3'-end of a polynucleotide ligation product that was previously obtained by ligation of two or more simple target probes that were annealed to a target sequence.
  • This first ligation splint oligo has a 3'-sequence that is complementary to the 3'-end ofthe target sequence and a second adjacent 5'-sequence that is complementary to the 5 '-end of a the 5 '-phosphorylated sense promoter sequence ofthe open circle probe.
  • the second ligation splint oligo has a 5'-sequence that is complementary to the 5 '-end ofthe target sequence and a second adjacent 3'-sequence that is complementary to the 3'-end ofthe open circle probe.
  • [00472] c. contacting the target probes annealed to the target nucleic acid sequence with a ligase under ligation conditions so as to obtain a linear ligation product; [00473] d. denaturing the ligation product from the target nucleic acid sequence; [00474] e. providing an open circle probe, wherein the 5 '-end portion ofthe open circle probe comprises a 5'-phosphate group and a sense promoter sequence for a double-sfranded franscription promoter that is recognized by a cognate RNA polymerase; [00475] f.
  • a first ligation splint comprising a 5'-end unphosphorylated portion that is complementary to the 5'-end portion ofthe open circle probe and a 3 '-end portion that is complementary to the 3 -end portion ofthe ligation product;
  • Still another embodiment ofthe invention comprises a method for detecting a target nucleic acid sequence, said method comprising:
  • [00483] a. providing at least two simple target probes comprising at least two target- complementary sequences, wherein the target probes comprise a 5 '-phosphate and are adjacent when annealed on the target sequence, and wherein a first simple target probe is complementary to the 5'-end ofthe target nucleic acid and a second simple target probe is complementary to the 3 '-end ofthe target nucleic acid sequence;
  • e providing an open circle probe, wherein the 5'-end portion ofthe open circle probe comprises a 5'-phosphate group and a sequence for single-sfranded franscription promoter or pseudopromoter that is recognized by a cognate RNA polymerase;
  • f providing a first ligation splint comprising a 5'-end unphosphorylated portion that is complementary to the 5'-end portion ofthe open circle probe and a 3'-end portion that is complementary to the 3 '-end portion ofthe ligation product;
  • the linear ligation product obtained in step (c) ofthe embodiments immediately above are ligated to an oligonucleotide comprising a sense promoter sequence for a double-stranded promoter or sequence for a single-sfranded promoter for a cognate RNA polymerase, thereby generating linear franscription subsfrates of the invention, meaning, for example, that simple target probes can be used without using a promoter target probe, and/or a signal target probe, and then, the resulting ligation product comprising the target-complementary sequence can be joined to a suitable sense promoter and a signal sequence, if used, by means of ligation splints and a ligase under ligation conditions.
  • Ligases that can be used to ligate suitable ends that are annealed to a ligation splint comprising DNA include, but are not limited to, Ampligase® DNA Ligase (EPICENTRE Technologies, Madison, WI), Tth DNA ligase, Tfl DNA ligase, Tsc DNA ligase (Prokaria, Ltd., Reykjavik, Iceland), or T4 DNA ligase. These ligases can be used for both intermolecular and intramolecular ligations when a ligation splint comprising DNA is used to bring the respective ends adjacent to each other.
  • T4 DNA ligase can be used to join the ends that are annealed to the ligation splint.
  • simple target probes that anneal adjacently on a target sequence are ligated, then denatured from the target sequence, then ligated to an oligo comprising a sense promoter sequence using a ligation splint that is complementary to the most 3'-end ofthe ligation product comprising the target-complementary target probes and to the 5'- end of a sense promoter sequence, and then, finally circularized by non-homologous intramolecular ligation of a 5'- ⁇ hosphorylated end with a 3'-hydroxyl end, and, if the promoter sequence comprises a sense promoter sequence for a double-sfranded promoter, annealed to an anti-sense promoter oligo to obtain a circular franscription subsfrate.
  • Circularization of a linear single-sfranded DNA without a ligation splint can be carried out using ThermoPhageTM RNA Ligase II (Prokaria, Ltd., Reykjavik, Iceland).
  • a reason to circularize a linear ligation product is to obtain a circular transcription subsfrate for more efficient transcription by a rolling circle transcription mechanism, rather than by linear transcription.
  • This embodiment is used only if steps are taken to assure that only ligation products derived from the target-complementary sequences that were ligated in the presence ofthe target sequence are circularized by the ligase that catalyzes non-homologous ligation, or that the other non-target-dependent franscription products will not be detected in the assay or method.
  • the present inventors' search for methods to detect a target analyte comprising a target nucleic acid sequence led, unexpectedly, to the to a novel concept for a method for sfrand displacement reverse transcription, which method provides unique possibilities for amplifying a target sequence and/or a signal sequence under certain conditions that are discussed below.
  • the method is useful in conjunction with other methods that utilize an RNA polymerase that can synthesize RNA using said ssDNA franscription substrates, such as, but not limited to the target- dependent transcription assays and methods ofthe present invention described herein.
  • Methods for strand displacement amplification of linear and circular ssDNA templates are well known in the art.
  • strand displacement amplification methods are disclosed in PCT Patent Publication Nos. WO 02/16639; WO 00/56877; and AU 00/29742; of Takara Shuzo Company; U.S. Patent Nos. 5,523,204; 5,536,649; 5,624,825; 5,631,147; 5,648,211; 5,733,752; 5,744,311; 5,756,702; and 5,916,779 of Becton Dickinson and Company; U.S. Patent Nos. 6,238,868; 6,309,833; and 6,326,173 of Nanogen Becton Dickinson Partnership; U.S. Patent Nos.
  • the methods for sfrand displacement amplification of linear templates in the art use some kind of process to digest a sequence region at or near the 5 '-end of a replicating second-strand cDNA in order to liberate at least a portion of the primer binding site on the DNA template so that another primer can anneal to the template and initiate DNA synthesis, which results in displacement ofthe last-synthesized DNA sfrand.
  • 6,251,639 of Kurn use a composite primer having a 5'- portion comprising ribonucleotides and a 3 '-portion comprising deoxynucleotides, and then use RNase H to liberate the primer-binding site at the 5 '-end ofthe replicating DNA sfrand.
  • Rolling circle amplification (RCA), as disclosed in U.S. Patent Nos. 6,344,329; 6,210,884; 6,183,960; 5,854,033; 6,329,150; 6,143,495; 6,316,229; 6,287,824; all of which are inco ⁇ orated herein by reference, including references therein, involve strand-displacement DNA polymerization using ssDNA templates.
  • another embodiment ofthe present invention comprises a method for amplifying a target nucleic acid comprising a linear single-sfranded RNA (ssRNA) by strand displacement reverse transcription, said method comprising: [00501 ] 1. providing a reaction mixture comprising:
  • each said primer comprises a sequence that is complementary to a sequence in said ssRNA; [00505] 2.
  • multiple primers are not required for strand-displacement reverse transcription of circular ssRNA templates because, under strand- displacement conditions, synthesis of first-strand cDNA proceeds around and around the circular ssRNA template, continually displacing first-strand cDNA synthesized during the previous round of reverse transcription, and generating a first-sfrand cDNA multimer comprising multiple tandem copies of a first-strand cDNA oligomer, each of which is complementary to one copy of said circular ssRNA molecule.
  • multiple primers are not required for circular ssRNA templates, the use of multiple primers is prefened in some embodiments in order to increase the rate of first-strand cDNA synthesis. Multiple primers are increasingly preferred as the size ofthe circular ssRNA template increases.
  • another embodiment ofthe present invention comprises a method for amplifying a target nucleic acid comprising a circular single-sfranded RNA (ssRNA) by sfrand displacement reverse transcription, said method comprising: [00509] 1. providing a reaction mixture comprising:
  • c at least one, and optionally multiple, oligonucleotide primers, wherein at least the 3 '-portion of each said primer comprises a sequence that is complementary to a sequence in said ssRNA;
  • [00513] 2. contacting said reaction mixture from step 1 above with a sample comprising a target nucleic acid comprising a circular ssRNA, wherein said reaction mixture containing said sample is maintained at a temperature wherein said reverse transcriptase, and optionally said single-strand binding protein, are optimally active in combination for strand-displacement reverse transcription and wherein said reverse transcription primers anneal to said target sequence, if present, with specificity, and wherein said temperature of said reaction mixture is maintained for a time sufficient to permit synthesis of first-sfrand cDNA reverse transcription products complementary to said target nucleic acid, if present in said sample; and [00514] 3. obtaining first-strand cDNA multirners comprising multiple tandem copies of a first-strand cDNA oligomer, each of which is complementary to one copy of said circular ssRNA target nucleic acid template.
  • Strand-displacement reverse transcription can be used to generate multiple copies of first-strand cDNA for use in methods and assays such as, but not limited to the embodiment shown in Figure 9.
  • RNA transcript RNA transcript
  • tail RNA transcript
  • the use of a tail is prefened according to the present strand-displacement reverse franscription method to facilitate sfrand displacement.
  • some embodiments ofthe present method comprise synthesis of first-sfrand cDNA by strand-displacement reverse transcription, wherein one or more reverse transcription primers comprising tail sequences that are not complementary to the RNA template are used.
  • the oligonucleotides used as strand-displacement primers comprise deoxyribonucleotides.
  • the invention also comprises other embodiments in which oligoribonucleotides are used for strand-displacement primers in the various embodiments of strand-displacement reverse transcription. Still further, the invention also comprises the use of 2'-fluoro-containing modified oligoribonucleotides or "DuraScriptTM RNA," which can be made using the DuraScribeTM T7 Transcription Kit (EPICENTRE Technologies, Madison, WI, USA) or purchased from oligonucleotide companies such as Integrated DNA Technologies, Coralville, IA, in the various embodiments of strand- displacement reverse transcription. Strand-displacement primers comprising DuraScriptTM RNA are resistant to RNase A-type ribonucleases.
  • Primers for strand-displacement reverse transcription can comprise a specific sequence that is complementary to only one RNA sequence.
  • the multiple strand-displacement primers of a strand-displacement reverse franscription reaction ofthe present invention can also comprise random sequence primers, including but not limited to random hexamers, random octamers, random nonamers, random decamers, random dodecamers and the like, with the length based on considerations such as the temperature optimum ofthe reverse transcriptase and the Tm random sequence primer.
  • the primers can also prime synthesis of second-strand cDNA using first-strand cDNA as a template, and subsequently, can prime the synthesis of third, fourth and other cDNA strands, thereby resulting in additional amplification.
  • Random sequence primers are commercially available from oligonucleotide companies such as Integrated DNA Technologies, Coralville, IA.
  • the random sequence primers comprise alpha-thio internucleoside linkages, which are resistant to some exonucleases.
  • a biotin or other binding moiety is covalently attached to a nucleotide in the 5 '-portion of a reverse transcription primer used for strand-displacement reverse franscription.
  • the biotin or other binding moiety enables capture of first-sfrand cDNA obtained by strand-displacement reverse franscription.
  • Conditions for strand-displacement reverse franscription can be identified by performing assays that measure the ability of a reverse transcriptase to displace a labeled oligo having a 3'-dideoxy nucleotide, wherein said labeled oligo is annealed to an RNA template 3 '-of or downstream of an extending first-strand cDNA that is being synthesized from a primer that anneals to said RNA template 5 '-of the site to which said labeled oligo anneals.
  • different reverse transcriptases, different reaction temperatures that cover the range for which each particular reverse franscriptase is active, and other reaction conditions are varied systematically in order to identify conditions that result in strand displacement reverse transcription.
  • Strand-displacing reverse transcriptases that can be used include, but are not limited to RNaseH-Minus MMLV reverse transcriptase (SuperscriptTM reverse transcriptases from Invifrogen, Carlsbad, CA), IsoThermTM DNA Polymerase (EPICENTRE Technologies, Madison, WI), or BcaBESTTM DNA Polymerase (Takara Shuzo Co., Japan).
  • RNaseH-Minus MMLV reverse transcriptase SuperscriptTM reverse transcriptases from Invifrogen, Carlsbad, CA
  • IsoThermTM DNA Polymerase EPICENTRE Technologies, Madison, WI
  • BcaBESTTM DNA Polymerase Takara Shuzo Co., Japan.
  • One reverse franscription reaction condition that can increase displacement of first-strand cDNA and which is included as part ofthe present invention, is addition of a single-strand binding protein, such as, but not limited to EcoSSB Protein or an SSB Protein from a thermostable bacterium, such as Tth or Bs
  • Betaine can also be added to a reverse transcription reaction in order to increase strand displacement.
  • it is prefened in many embodiments to use a final concentration of about 0.25 M, about 0.5 M, about 1.0 M, about 1.5 M, about 2.0 M, about 2.5 M or between about 0.25 M and 2.5 M betaine (trimethylglycine) in DNA polymerase or reverse franscriptase reactions in order to decrease DNA polymerase stops and increase the specificity of reactions that use a DNA polymerase.
  • reaction conditions that result in strand-displacement reverse franscription will result in further amplification of a target sequence and/or a signal sequence in a target-dependent transcription assay or method ofthe present invention.
  • a target sequence in a target nucleic acid is present in a sample, the methods of the invention that use a bipartite target probe, as disclosed herein, can be used to generate a circular transcription substrate ofthe present invention.
  • This circular transcription subsfrate can be used as a substrate for rolling circle transcription by an RNA polymerase that binds to a promoter and synthesizes RNA therefrom in order to synthesize double-sfranded RNA (dsRNA) that can be used to silence a gene by RNA interference. That is the dsRNA is used as RNAi.
  • dsRNA for use as RNAi can be synthesized using the embodiment ofthe invention illustrated in Figure 9.
  • the target sequence comprises a target nucleic acid that is encoded by a pathogen or by an oncogene
  • the dsRNA can be a therapeutic composition.
  • a new circular franscription subsfrate is prepared for synthesis of dsRNA for use as RNAi, wherein each oligomer ofthe RNA multimer transcription product obtained using said circular transcription subsfrate for rolling circle franscription comprises a self-complementary double-sfranded hai ⁇ in structure with a non-complementary loop between the self-complementary regions, such that each oligomer corresponds to the desired RNAi and the loop structure.
  • said circular transcription subsfrate is designed so that said RNA oligomers can be cleaved from the RNA multimer obtained from rolling circle franscription, for example, using a ribozyme or an RNase H and DNA oligo complementary to the cleavage site.
  • Prefened RNA polymerases for rolling circle franscription comprise T7 RNAP,
  • T3 RNAP, or SP6 RNAP or mutant enzymes such as but not limited to T7 RNAP Y639F, T3 RNAP Y573F or SP6 RNAP Y631F mutant enzymes (Sousa et al., U.S. Patent No. 5,849,546).
  • some embodiments of this aspect ofthe invention use an N4 mini-vRNAP enzyme, such as but not limited to an N4 mini-vRNAP Y678F mutant enzyme (U.S. Patent Application No.
  • RNAi RNA multimers comprising double- stranded hafrpins for use in RNAi.
  • Most prefened embodiments of of this aspect ofthe invention use one ofthe T7-type RNAPs or the N4 mini-vRNAP Y678F mutant enzyme to synthesize RNA containing 2'-fluoro-pyrimidine nucleotides by using 2'-fluoro-dCTP and 2'- fluoro-dUTP, in addition to ATP and GTP in the rolling circle transcription reaction.
  • RNA molecules that contain 2'-F-dCMP and 2'-F-dUMP are resistant to RNase A-type ribonucleases (Sousa et al, U.S. Patent No. 5,849,546), included herein by reference.
  • Capodici et al, (J. Immunology, 169: 5196-5201, 2002 showed that 2'-fluoro-containing dsRNA molecules made using the DuraScribeTM Transcription Kit (EPICENTRE Technologies, Madison, WI, USA) did not require transfection reagents for delivery into cells, even in the presence of serum.
  • Kakiuchi et al. J. Biol.
  • Target-Dependent Transcription Using Monopartite Target Probes Comprising a T7 Promoter to Detect the Human ⁇ -glohin Gene Sequence in Which a Single Nucleotide Mutation Results in Sickle Cell Anemia
  • Monopartite target probes were designed to anneal to the gene encoding human jS-globin.
  • the ligation junction ofthe adjacent probes when annealed to the denatured globin gene is the site of a single-base difference responsible for the sickle-cell phenotype (an A to T fransversion).
  • the globin promoter target probe and the globin signal target probe should only ligate when annealed to the wild-type globin allele, but not when the ligation junction is annealed to a target nucleotide comprising a single-base mismatch that results in the sickle-cell phenotype.
  • Oligonucleotide target probes and an anti-sense promoter oligo were obtained from Integrated DNA Technologies, Coralville, IA.
  • the target-complementary sequences are underlined.
  • the T7 sense promoter sequence is in italics.
  • the remaining portion ofthe globin signal target probe serves as a signal sequence.
  • A. Globin Promoter Target Probe (37 nucleotides, 15 target-complementary nucleotides):
  • a promoter target probe and signal target probe for detection of a beta-globin gene sequence were designed with a goal to be optimal for both hybridization specificity and thermostable ligase activity under hybridization and ligation reaction conditions.
  • the target-complementary portions ofthe target probes were long enough to hybridize preferentially to the target sequence and not elsewhere in the human genome at a hybridization temperature that would still provide sufficient thermostable ligase activity.
  • the probe sequences were compared to the Genbank database to verify homology to the targets and to identify any secondary targets.
  • the promoter target probe had 15 target-complementary bases, 17 bases of T7 sense promoter sequence and +1 nucleotide, and 4 additional non-homologous bases upstream ofthe promoter for improved transcription, and the 5' end was phosphorylated.
  • the signal target probe contained 25 target-complementary bases at the 3' end and 25 bases of a signal sequence at the 5' end for detection ofthe franscription product.
  • the globin target probes were incubated with subcloned plasmid DNA containing the wild-type globin gene sequence.
  • Target probes were annealed and ligated to the target sequence as follows: From 10-400 nanograms of plasmid DNA comprising the target sequence were denatured and hybridized to 2 to 50 picomoles of each target probe in 20 mM Tris-HCl (pH 8.3 at 25°C), 25 mM KCl, 10 mM MgCl 2 , 0.5 mM NAD, 0.01% Triton X-100 at 94°C for 2 minutes.
  • Target probes annealed to the target sequence were ligated using 100 Units of Ampligase ® Thermostable Ligase (EPICENTRE Technologies, Madison, WI) by thermocycling for 20 to 50 cycles of 94°C for 30 seconds and 40°C for 4 minutes. Then, in order to generate a double-sfranded T7 promoter, an excess ofthe anti-sense promoter oligo (5-50 picomoles) was annealed to the unpurified ligation products by heating to 94°C for 2 minutes and slow cooling to room temperature.
  • Ampligase ® Thermostable Ligase EPICENTRE Technologies, Madison, WI
  • Transcription reactions were analyzed as follows: 10-25 % ofthe ligation reaction was used as template in the transcription reaction. Thus, the equivalent of 0.5 to 5 picomoles of starting target probe from the ligation reaction was used as template in an AmpliScribeTM Tl -FlashTM in vitro transcription reaction (EPICENTRE Technologies, Madison, WI), without purification ofthe linear transcription subsfrate.
  • the 20 ul reaction contained 9 mM each NTP, IX AmpliScribe Tl -Flash buffer, 10 mM DTT, and AmpliScribe Tl -Flash Enzyme Mix. Reactions were incubated at 37°C for 2 hours.
  • the transcription reactions were treated with 2 Units DNase I, for 30 minutes at 37°C. Samples were heat denatured in formamide loading buffer and were analyzed by denaturing polyacrylamide gel electrophoresis using 15% gels, 6 M urea in IX TBE.
  • RNA franscription product was observed only in reactions in which the globin target probes were incubated under hybridization and ligation conditions in the presence ofthe wild-type -globin sequence. No franscription product was observed in the absence ofthe wild-type ⁇ -globin sequence, in the absence of ligase, or in the presence of only one target probe.
  • Monopartite Target Probes Comprising a T7 Promoter to Detect Human Papilloma Virus (HPV) Gene Sequences
  • HPV Human papilloma virus
  • HPV is believed to be responsible for human diseases including cervical cancer and warts. Certain strains appear to be related to higher cancer risk than others. Detection ofthe presence ofthe virus and the strain ofthe virus is useful for research and diagnostics.
  • Target-dependent transcription reactions were performed using monopartite target probes comprising a T7 promoter in order to make an initial evaluation ofthe sensitivity and specificity of this method for detection of an HPV DNA sequence.
  • Oligonucleotide target probes and an anti-sense promoter oligo were obtained from Integrated DNA Technologies, Coralville, IA. The target-complementary sequences are underlined. The T7 sense promoter sequence is in italics. The remaining portion ofthe HPV signal target probe serves as a signal sequence.
  • HPV Promoter Target Probe 38 nucleotides, 16 target-complementary nucleotides:
  • HPV Signal Target Probe 50 nucleotides; 25 target-complementary nucleotides:
  • a promoter target probe and signal target probe for detection ofthe major capsid protem LI gene of HPV type 16, commonly found in cervical cancer specimens, were designed with a goal to be optimal for both hybridization specificity and thermostable ligase ligation activity under hybridization and ligation reaction conditions.
  • the target-complementary portions ofthe target probes were long enough to hybridize preferentially to the target sequence and not elsewhere in the human genome at a hybridization temperature that would still provide sufficient thermostable ligase activity.
  • the probe sequences were compared to the Genbank database to verify homology to the targets and to identify any secondary targets.
  • the promoter target probe had 16 target-complementary bases, 18 bases of T7 sense promoter sequence and +1 nucleotide, and 4 additional non-homologous bases upstream ofthe promoter for improved transcription, and the 5' end was phosphorylated.
  • the signal target probe contained 25 target-complementary bases at the 3' end and 25 bases of a signal sequence at the 5' end for detection ofthe transcription product.
  • HPV target probes were incubated with denatured HPV16 LI gene PCR product containing 456 bases of major capsid protein LI sequence.Target probes were annealed and ligated to the target sequence as follows: From 10-400 nanograms of PCR product comprising the target sequence were denatured and hybridized to 2 to 50 picomoles of each target probe in 20 mM Tris-HCl (pH 8.3 at 25°C), 25 mM KCl, 10 mM MgCl 2 , 0.5 mM NAD, 0.01% Triton X-100 at 94°C for 2 minutes.
  • Target probes annealed to the target sequence were ligated using 100 Units of Ampligase ® Thermostable Ligase (EPICENTRE Technologies, Madison, WI) by thermocycling for 20 to 50 cycles of 94°C for 30 seconds and 40°C for 4 minutes. Then, in order to generate a double-sfranded T7 promoter, an excess ofthe anti-sense promoter oligo (5-50 picomoles) was annealed to the unpurified ligation products by heating to 94°C for 2 minutes and slow cooling to room temperature.
  • Ampligase ® Thermostable Ligase EPICENTRE Technologies, Madison, WI
  • Transcription reactions were analyzed as follows: 10-25 % ofthe ligation reaction was used as template in the transcription reaction. Thus, the equivalent of 0.5 to 5 picomoles of starting target probe from the ligation reaction was used as template in an AmpliScribeTM Tl -Flash ' TM in vitro transcription reaction (EPICENTRE Technologies, Madison, WI), without purification ofthe linear transcription substrate.
  • the 20 ul reaction contained 9 mM each NTP, IX AmpliScribe Tl -Flash buffer, 10 mM DTT, and AmpliScribe Tl -Flash Enzyme Mix. Reactions were incubated at 37°C for 2 hours.
  • the transcription reactions were treated with 2 Units DNase I, for 30 minutes at 37°C. Samples were heat denatured in formamide loading buffer and were analyzed by denaturing polyacrylamide gel elecfrophoresis using 15% gels, 6 M urea in IX TBE.
  • annealing ofthe T7 anti-sense promoter oligo to the globin promoter target probe yields only a 18-nucleotide transcript, and no signal sequence would be obtained.
  • the 68- nucleotide transcript was specifically transcribed only in the reactions containing full-length ligation products. No 68-nucleotide transcription products were observed without the ligase or in the absence of either HPV target probe. The 68-nucleotide transcription product could be detected at all levels of target probes tested between 2 and 20 picomoles per reaction.
  • HPV 1 DNA in mock-patient DNA samples was also performed with mixtures of HP VI 6 LI gene PCR product with human genomic DNA.
  • the HPV target probes ligation product and resulting RNA transcript were not produced with human genomic DNA alone, but were produced with samples containing both human genomic DNA and different dilutions of HPV16 DNA.
  • the transcript from the template-dependent ligation probe was detectable by gel electrophoresis when as little as 33 femtomoles of HPV 16 target sequence was present in 100 ng of human genomic DNA. This implies that the currently described target-dependent transcription method could be used to detect viral DNA in patient samples.
  • the sensitivity ofthe reaction could be increased still further by using an amplifiable signal sequence in the HPV signal target probe. That this method detected the presence of a target sequence in a mixed DNA population indicates that DNA viruses such as human papilloma virus (HPV) can be detected in complex human DNA samples using monopartite target probes for target-dependent franscription.
  • HPV human papill
  • Each oligonucleotides (50 picomoles), comprising a sense P2 promoter sequence
  • RNA Ligase II (Prokaria, Rejkjavik, Iceland, #Rligl22) in IX ThermoPhage RNA Ligase II Buffer comprising 50 mM MOPS, pH 7.5, 5 mM MgCl 2 , and 10 mM KCl. Then, linear oligos were removed by digestion with Exonuclease I (EPICENTRE Technologies, Madison, WI), the Exo I was heat-inactiv
  • the resulting mini-vRNAP franscription products were then analyzed by electrophoresis in a 1% agarose gel containing 0.22 M formaldehyde. Transcription products, including products having a length many-fold greater than the starting oligonucleotide, were observed on the the gel using the franscription subsfrate having a sense P2 promoter sequence, indicating efficient rolling circle transcription. No transcription products were observed if the oligo did not contain a P2 promoter, if an anti-sense sequence to the P2 promoter was used instead ofthe sense P2 promoter, or if an unligated linear oligo with a sense P2 promoter was used.
  • Bipartite Target Probes Comprising a P2 Promoter to Detect the Human ⁇ -globin Gene Sequence in Which a Single Nucleotide Mutation Results in Sickle Cell Anemia
  • Bipartite target probes were designed to anneal to the gene encoding human hemoglobin ⁇ chain.
  • the ligation junction ofthe adjacent probes when annealed to the denatured globin gene is the site of a single-base difference responsible for the sickle-cell phenotype (an A to T fransversion leading to Glu- Val change in the /5-globin).
  • the probe can be circularized by DNA ligase only when annealed to the wild-type globin allele, but not when the ligation junction is annealed to a target nucleotide comprising a single-base mismatch that results in the sickle-cell phenotype.
  • Oligonucleotide target probes were obtained from Integrated DNA Technologies,
  • All human / 3-globin bipartite target probes consisted of two target-complementary arms in the 5' and 3' terminal regions connected by a spacer of a specific size that contained P2 promoter sequence, as well as (optional) binding sites for amplification primers, restriction sites, signal sequences, etc.
  • the 5' arm length was from 11 to 18 nucleotides and was designed to anneal immediately upsfream ofthe single-base mismatch that results in the sickle-cell phenotype.
  • the 3' arm was from 14 to 20 nucleotides long and was complementary to the region immediately downstream of this mutation.
  • the 3'-terminal base was complementary to the nucleotide that differed in the wild type and mutant alleles ofthe / 3-globin gene. This was done to improve allele discrimination, since base mismatches at the 3' terminus are more inhibitory to ligation than those at 5' terminus (Luo et al., Nucleic Acids Res., 24:3071-3078, 1996).
  • the length ofthe spacer region should allow circularization ofthe oligo while its 5' and 3' terminal arms are annealed to the target and cannot be shorter then 1.26- times the combined length of target-complementary arms.
  • the target-complementary sequences are underlined.
  • the P2 promoter hai ⁇ in sequence is in italics.
  • Bipartite target probes for detection of a beta-globin gene sequence were designed with a goal to be optimal for (i) target recognition specificity and thermostable ligase activity under hybridization and ligation reaction conditions and (ii) for N4 mini-vRNAP- catalyzed rolling circle transcription.
  • the probe was designed so that the P2 promoter hai ⁇ in was the only stable secondary structure at 37°C, the target-complementary portions of the target probes were long enough to hybridize preferentially to the target sequence at a hybridization temperature that would still provide sufficient thermostable ligase activity.
  • the overall length ofthe probe was kept to a minimum (under 100 nucleotides) to ensure efficient rolling circle transcription.
  • the -globin bipartite target probes were incubated with subcloned plasmid DNA containing the wild-type
  • Target probes were annealed and ligated to the target sequence as follows: 2.5 micrograms of plasmid DNA comprising the target sequence were denatured and hybridized to 2 to 50 picomoles of each target probe in 20 mM Tris-HCl (pH 8.3 at 25°C), 25 mM KCl, 10 mM MgCl 2 , 0.5 mM NAD, 0.01% Triton X-100 at 94°C for 1.5 minutes in the total volume of 50 ul.
  • Target probes annealed to the target sequence were ligated using 50 Units of Ampligase ® Thermostable Ligase (EPICENTRE Technologies, Madison, WI) by thermocychng for 20 to 50 cycles of 94°C for 30 seconds and 40°C for 6 minutes. The unligated probe was then removed by digestion with 40 units of E. coli Exonuclease I (EPICENTRE) for 30 minutes at 37°C. Ligation reactions were ethanol-precipitated or used directly as subsfrates for the N4 mini- vRNAP transcription.
  • Transcription reactions were analyzed as follows: 10-25 % ofthe ligation reaction was used as template in the franscription reaction.
  • the 20 ul reactions contained 1 mM each NTP, 1 mM DTT, 5 uM EcoSSB Protein (EPICENTRE), 1 U/ul RNasin (Promega, Fitchburg, WI), and 8 pmol N4 mim-vRNAP (EPICENTRE) in lx transcription buffer comprising 40 mM Tris HC1, pH 7.5, 10 mM NaCl, 6 mM MgCl 2 , and 1 mM spermidine. Reactions were incubated at 37°C for 2 to 6 hours.
  • the transcription reactions were freated with 2 Units DNAse I for 30 minutes at 37°C. Samples were heat denatured in formamide loading buffer with 0.1% SDS and were analyzed by denaturing 1% agarose gel electrophoresis in IX TAE buffer.
  • a bipartite probe oligo circularizes and serves as efficient template for N4 mini-vRNAP-catalyzed rolling circle transcription, yielding high molecular weight RNA products.
  • the unligated linear probe yields only a 11-18-nucleotide transcript (and no signal sequence would be obtained).
  • high molecular weight RNA transcription products were observed only in reactions in which the wild type -globin target probes were incubated under hybridization and ligation conditions in the presence ofthe wild-type -globin sequence. No high molecular weight transcription product was observed in the absence ofthe wild-type 5-globin sequence, in the absence of ligase, or in the presence of only one target probe.
  • RNA template for strand displacement reverse transcription was first obtained by in vitro transcription of a PCR product that contained a T7 promoter sequence that was inco ⁇ orated using a promoter sequence-containing PCR primer.
  • the PCR product was in turn obtained by amplifying a linearized plasmid using the following two primers:
  • the PCR reaction mixture was prepared with a final volume of 50 ul and contained 1 ng ofthe linearized plasmid, 12.5 pmoles of each ofthe above two primers, 25 ul of 2x High Fidelity Long PCR PreMix 4 (EPICENTRE Technologies, Madison, WI) and 2.5 Units ofMasterAmpTM Extra Long DNA Polymerase Mix (EPICENTRE).
  • the PCR reaction mixture was heated to 95° C for 1 min and then subjected to 35 reaction cycles of 95 °C for 45 sec, 50 °C for 45 sec, and 70 °C for 3 min.
  • the PCR products were extracted and ethanol-precipitated using standard techniques, and resuspended in 25 ul of 10 mM Tris.HCl (pH 8.0), 1 mM EDTA (TE).
  • TE 1 mM EDTA
  • the resulting PCR product was then used to prepare a 1-Kb linear RNA transcript as follows: In vitro franscription with was performed using 5 ul of PCR product DNA as a template and the reagents supplied with the AmpliscribeTM T7 FlashTM Transcription Kit (EPICENTRE). The transcription products were treated with DNAse I, extracted and ethanol- precipitated using standard techniques, and resuspended in TE buffer at a concentration of 1.5 ug/ul (4.5 pmoles/ul).
  • the 1-Kb linear RNA transcript was then freated with tobacco acid pyrophosphatase (TAP) and T4 RNA ligase in order to obtain a 1-Kb circular RNA transcript as follows: The 5 '-end ofthe RNA transcript (20 pmoles in 20 ul) containing pppG was converted to pG using 20 Units of Tobacco Acid Pyrophosphatase (EPICENTRE) at 37 °C for 45 minutes.
  • TEPICENTRE tobacco acid pyrophosphatase
  • RNA from this reaction mix (2 ul) was incubated in 10 ul of a ligation mixture containing 33 mM Tris acetate (pH 7.8), 66 mM Potassium acetate, 10 mM magnesium acetate, 1 mM DTT and 5.0 U of T4 RNA ligase (EPICENTRE) at 37 °C for 1 hour.
  • the 1-Kb circular RNA transcript or the 1-Kb linear RNA transcript was incubated under reverse transcription reaction conditions as follows: The reverse transcription was performed by preparing a reaction mixture containing 2.0 ul ofthe ligation mix, 50 mM Tris-HCl (pH 9.0), 12.5 mM NaCl, 20 mM (NH 4 ) 2 S0 4 , IX MasterAMPTM PCR Enhancer with betaine (EPICENTRE), 250 uM of each of dATP, dCTP, dGTP and dTTP, 200 Units of IsothermTM DNA Polymerase (EPICENTRE), and 12.5 pmoles of a strand-displacement primer comprising the same RNAI 000 Primer as used above for PCR. The reaction mixture was incubated at 50 °C for 90 minutes. The products ofthe rolling circle reverse transcription reaction were analyzed on a 1.0 % formaldehyde agarose gel and visualized by staining with SYBR® gold (Molecular

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Abstract

La présente invention concerne de nouvelles méthodes, compositions et trousses comprenant des sondes cibles monopartites ou bipartites et une ARN polymérase destinées à détecter et quantifier des substances à analyser comprenant une ou plusieurs séquences d'acide nucléique, y compris des séquences cibles qui diffèrent seulement d'un nucléotide; ou à détecter et quantifier des substances à analyser exemptes d'acide nucléique, par détection d'une étiquette de séquence cible associée à une substance de liaison aux substances à analyser. Le procédé de l'invention, appelé 'transcription dépendante de la cible', comprend un traitement de renaturation, un processus de ligature d'ADN, un processus de transcription, et un processus de détection. L'invention concerne en outre de nouvelles méthodes, compositions et trousses destinées à amplifier l'ARN, y compris par transcription inverse par déplacement de brins et transcription inverse par cercle roulant.
EP03814301A 2002-12-23 2003-12-23 Transcription dependante de la cible Withdrawn EP1583840A4 (fr)

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CN106801050B (zh) * 2017-02-20 2020-01-14 广州永诺生物科技有限公司 一种环状rna高通量测序文库的构建方法及其试剂盒
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WO2002044339A2 (fr) * 2000-12-01 2002-06-06 Zhang David Y Procede d'amplification de l'acide nucleique

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US5876924A (en) * 1994-06-22 1999-03-02 Mount Sinai School Of Medicine Nucleic acid amplification method hybridization signal amplification method (HSAM)
EP1098996A1 (fr) * 1998-07-20 2001-05-16 Yale University Procede pour detecter des acides nucleiques au moyen de ligature a mediation par cibles d'amorces bipartites

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WO2002044339A2 (fr) * 2000-12-01 2002-06-06 Zhang David Y Procede d'amplification de l'acide nucleique

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Title
OHMICHI TATSUO ET AL: "Efficient bacterial transcription of DNA nanocircle vectors with optimized single-stranded promoters" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 99, no. 1, 8 January 2002 (2002-01-08), pages 54-59, XP002497227 ISSN: 0027-8424 *
See also references of WO2004058989A2 *

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EP1583840A4 (fr) 2009-03-18

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