WO2014158628A1

WO2014158628A1 - Compositions and methods for analysis of nucleic acid molecules

Info

Publication number: WO2014158628A1
Application number: PCT/US2014/018887
Authority: WO
Inventors: Jeff G. Hall
Original assignee: Hologic, Inc.
Priority date: 2013-03-14
Filing date: 2014-02-27
Publication date: 2014-10-02

Abstract

The present invention relates to assays for the detection of nucleic acid. In accordance with the present invention, methods, reaction mixtures, compositions and kits are provided for the production of specific nucleic acid molecules called priming oligonucleotides, for use in assays for the detection, characterization, and/or quantitation of nucleic acid target sequences.

Description

COMPOSITIONS AND METHODS FOR

ANALYSIS OF NUCLEIC ACID MOLECULES

The present application claims priority to U.S. Provisional Application Serial No. 61/784,273, filed March 14, 2013, which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention provides systems, methods and kits for generating detectable signal in nucleic acid detection reactions. In some embodiments, the detectable signal is amplified using a reaction design in which the products of an initial signal generating reaction precipitate the production of additional detectable signals in a cascading fashion. The detection assays employ nucleic acid synthesis to form a substrate for modification by a nucleic acid modifying enzyme (e.g., cleavage by a FEN-1 endonuclease).

BACKGROUND

The detection, characterization, and quantification of nucleic acids play important roles in the fields of biology and medicine. For example, quantification of nucleic acid is important in cancer diagnosis and prognosis and viral diagnosis and judgments of therapeutic effects.

In many reactions based on detection of cleaved probe oligonucleotides, e.g., TAQMAN and INVADER assay reactions, impure synthetic oligonucleotides contribute to background signal that can compromise the sensitivity and/or specificity of the assays. In particular, chemically synthesized oligonucleotides prepared for use, e.g., as probes in nucleic acid detection reactions may contain truncated failure sequences (partial reaction products). Furthermore, post-synthetic treatments such as high-temperature deprotection protocols can also damage synthetic oligonucleotides, causing loss of base moieties (e.g., depurination) or breakage of the oligonucleotide strands (e.g., at abasic sites). See e.g., US 7,582,436, incorporated herein by reference for all purposes.

What is needed are relatively simple and inexpensive methods for detecting and quantitating nucleic acids in a target-specific fashion that generate signal in a manner that is not influenced by oligonucleotide fragments found in synthetic oligonucleotide preparations.

SUMMARY OF THE INVENTION

The present invention provides systems, methods and kits for generating detectable signal in nucleic acid detection reactions. In some embodiments, the detectable signal is amplified using a reaction design in which the products of an initial signal generating reaction precipitate the production of additional detectable signals in a cascading fashion.

In some embodiments, provided herein are methods of detecting a target nucleic acid, comprising:

a) in a reaction comprising a target nucleic acid, forming a priming oligonucleotide comprising a distinctive 3' end, wherein formation of said priming oligonucleotide is indicative of the presence of said target nucleic acid;

b) hybridizing said priming molecule to an oligonucleotide template and forming a detectable structure;

c) detecting said detectable structure.

In some embodiments, forming a priming oligonucleotide comprises extending a primer on the target nucleic acid with a polymerase to form an extended primer having an extension region.

In some embodiments, the method further comprises cleaving the extended primer in the extension region to form the priming oligonucleotide.

In some embodiments, forming the detectable structure comprises extending the priming oligonucleotide to form an extended priming oligonucleotide having an extension region.

The extending of the primer and/or the priming oligonucleotide are not limited to any particular means of extension. For example, in some embodiments extension is with a polymerase while in some embodiments, a ligase may be used. In certain preferred embodiments, the primer and/or the priming oligonucleotide are extended with a template dependent polymerase. In particularly preferred embodiments, the polymerase is a DNA polymerase. In some embodiments, the polymerase is thermostable.

In some embodiments, a detectable structure formed in the method comprises a cleavage structure, e.g., cleavable by structure-specific nuclease. In certain preferred embodiments, the cleavage structure is an invasive cleavage structure. In some embodiments, detecting comprises cleaving the cleavage structure and in certain preferred embodiments, cleaving the cleavage structure comprises cleaving the extended priming oligonucleotide. In certain embodiments, cleaving the extended priming oligonucleotide comprising cleaving the extension region off said extended priming oligonucleotide to release a priming oligonucleotide. In preferred embodiments, the released priming oligonucleotide has the same sequence as the original priming oligonucleotide, i.e., the cleavage removes the extension region and restores the priming oligonucleotide to its un-extended form.

In some embodiments, the oligonucleotide template is configured to participate in formation of a detectable structure. For example, in some embodiments, the

oligonucleotide template is an extension/cleavage template oligonucleotide having a 3' portion, such that said invasive cleavage structure forms when the 3' portion of the extension/cleavage template displaces one or more nucleotides of a duplex between the extended priming oligonucleotide and the extension/cleavage template.

In some embodiments, cleaving the extended priming oligonucleotide produces a distinctive detectable molecule, e.g., a released "arm" or "flap" oligonucleotide. The arm or flap oligonucleotide cleavage product may be detected by any number of suitable methods for detecting a small nucleic acid. For example, in some embodiments, detecting comprises hybridizing the released arm oligonucleotide to a FRET cassette to form a detectable structure, such as an invasive cleavage structure. In preferred embodiments, the FRET cassette is cleaved to generate detectable fluorescence.

Detection of the detectable structures is not limited to any particular means of detection. For example, detecting may comprises detection one or of: detection of fluorescence, mass, fluorescence energy transfer, radioactivity, luminescence, phosphorescence, fluorescence polarization, and charge, or combinations thereof. In embodiments comprising cleavage, in preferred configurations, the reaction comprises a 5' nuclease, e.g., a FEN-1 endonuclease, preferably from an archaeal species. Many different polymerases find application in the methods provided herein. For example, RNA polymerases, DNA polymerases, ligases, etc. In certain embodiments, the polymerase comprises a DNA polymerase from Bacillus stearothermophilus .

The target nucleic acid is not limited to a particular type. For example, in some embodiments, the target nucleic acid target nucleic acid is selected from the group consisting of DNA and RNA. In some embodiments, the target nucleic acid is isolated from a sample, while in some embodiments, the nucleic acid is synthetic, or is, e.g., an amplified product, e.g., a polymerase chain reaction product. In still further

embodiments, the target nucleic acid is modified in vivo or in vitro. For example, nucleic acid may be methylated, or it may be bisulfite converted such that unmodified residues (e.g., unmethylated Cs) are converted (e.g., to uracils).

Some embodiments of the invention make use of conditions and reagents that facilitate particular behaviors of nucleic acids, e.g., strand invasion. For example, osmolytes such as betaine and/or trimethylamine N-oxide may facilitate strand invasion. In some embodiments, the reaction comprises an osmolyte.

In some embodiments, it is contemplated that a method of detecting a target nucleic acid, comprises incubating a target nucleic acid under conditions wherein:

i) a primer is extended on the target nucleic acid to form an extended primer; ii) the extended primer is modified to form a priming oligonucleotide comprising a distinctive 3' end;

iii) the priming oligonucleotide hybridizes to an extension/cleavage template oligonucleotide and is extended to form an extended priming oligonucleotide; iv) a snaking region of the extension/cleavage template oligonucleotide forms a cleavage structure comprising the extended priming oligonucleotide; and v) the extended priming oligonucleotide is cleaved to form a cleavage product. In certain preferred embodiments, the method further comprises detecting cleavage of the extended priming oligonucleotide. In some embodiments, the priming oligonucleotide is produced from the extended primer by cleaving. In certain preferred embodiments, the cleaving is by a structure specific nuclease, and in particularly preferred embodiments, the structure specific nuclease is a 5' nuclease, more preferably a FEN-1 endonuclease.

In some embodiments, the invention contemplates a composition for detecting a target nucleic acid, the composition comprising:

i) a priming oligonucleotide comprising a distinctive 3' end;

ii) an extension/cleavage template oligonucleotide comprising a region complementary to at least a portion of said priming oligonucleotide and a snaking region configured to form a cleavage structure when said priming oligonucleotide is extended by a template-dependent polymerase; iii) a polymerase activity; and

iv) a structure-specific nuclease activity. In some embodiments, the polymerase activity is provided as a DNA or R A polymerase and in particularly preferred embodiments, the polymerase activity is template-dependent.

In certain embodiments, the structure specific nuclease activity is a 5' nuclease activity, preferably a 5' nuclease activity. In certain preferred embodiments, the 5' nuclease activity is provided as FEN-1 endonuclease and/or a DNA polymerase. In some embodiments, the polymerase activity and said structure specific nuclease activity are provided as a single enzyme.

In some embodiments, the cleavage structure is a structure or employs reagents or approaches described in one or more of: U.S. Pat. Nos.: 7,312,033, 7,306,917, 7,297,780, 7,273,696, 7,256,020, 7,195,871, 7,150,982, 7,101,672, 7,087,381, 7,067,643, 7,060,436, 7,045,289, 7,011,944, 6,932,943, 6,913,881, 6,875,572, 6,872,816, 6,780,982, 6,780,585, 6,759,226, 6,709,819, 6,709,815, 6,706,471, 6,692,917, 6,673,616, 6,635,463, 6,562,611, 6,555,357, 6,458,535, 6,372,424, 6,358,691, 6,355,437, 6,348,314, 6,214,545, 6,194,149, 6,090,606, 6,090,543, 6,001,567, 5,994,069, 5,985,557, 5,888,780, 5,846,717, 5,843,669, 5,843,654, 5,837,450, 5,719,028, 5,614,402, and 5,541,311 and U.S. Publ. Nos.:

20080015349, 20080014124, 20070292856, 20070207455, 20070202517, 20070111200, 20070087345, 20070049745, 20060252032, 20060246475, 20060240452, 20060199202, 20060183207, 20060160074, 20060147955, 20060147938, 20050277138, 20050196750, 20050186588, 20050181435, 20050164177, 20050158716, 20050130179, 20050106596, 20050074788, 20050048527, 20040219576, 20040203035, 20040096874, 20040014067, 20030219784, 20030143535, 20030134349, 20030124526, 20030113237, 20030113236, 20030104470, 20030104378, 20030092039, 20030082544, 20030072689, 20020156255, 20020142454, and 20020128465, each of which is herein incorporated by reference in its entirety. These patents and published applications also describe enzymes, design, manufacture, and detection systems, and other components useful in the methods, compositions, and systems of the invention.

In some embodiments, the method produces a priming oligonucleotide having a defined composition region (DCR) and a distinct 3' end (D3E) region. In some embodiments, the DCR contains a sufficient number of nucleotides to permit specific binding to a complementary nucleic acid in a reaction comprising a mixture of complex nucleic acids, e.g., mRNA, genomic DNA, etc. In some embodiments, the DCR and the D3E together comprise fewer than about 11 (e.g., 10, 9, 8, 7, 6, etc.) nucleotides that are complementary to said ECT oligonucleotide. In some embodiments, one or more of the oligonucleotides in the reaction (e.g., the primer, the priming oligonucleotide, the ECT oligonucleotide, a secondary template oligonucleotide) contains one or more non-natural nucleotides, while in some embodiments, the primer, priming oligonucleotide, ECT oligonucleotide, FRET cassette and other oligonucleotides in a composition consist of naturally occurring nucleotides. In some embodiments, one or more of the ECT, a probe oligonucleotide and a FRET cassette have a moiety at the 3' end that prevents extension of the molecule(s) by a polymerase.

In some embodiments, the detecting step utilizes a detection oligonucleotide. For example, in some embodiments, the detecting of the cleaved priming oligonucleotide comprises associating one or more of said cleaved fragments with a synthetic detection oligonucleotide. In some embodiments, the synthetic detection oligonucleotide has a region of self-complementarity that forms a hairpin structure. In some embodiments, the synthetic detection oligonucleotide comprises a label (e.g., a fluorescent label). In some embodiments, the synthetic detection oligonucleotide further comprises a fluorescent quencher moiety. In some embodiments, the cleaved fragments are detected by extension of the cleaved fragments, using the detection oligonucleotide as a template. In some embodiments, the cleaved fragments are detected by ligation of the cleaved fragments to another molecule, using the detection oligonucleotide as a template. In some

embodiments, the cleaved fragments, when associated with the synthetic detection oligonucleotide, form a cleavage structure that is cleavable by the FEN-1 endonuclease. In some embodiments, the detecting comprises cleaving the cleavage structure (that comprises the synthetic detection oligonucleotide) to generate a detectable signal.

In some embodiments, an unknown target nucleic acid is analyzed in combination with a known synthetic control target nucleic acid, to, for example, determine an amount of the unknown target nucleic acid.

The present invention also provides compositions and systems containing one or more components useful, necessary, or sufficient for conducting any of the methods described herein. For example, in some embodiments, a composition comprises: a) a target nucleic acid; b) a primer; c) a polymerase; d) a FEN-1 endonuclease; and e) an extension/cleavage template (ECT) oligonucleotide comprising a portion complementary to a defined composition region (DCR) and universal oligonucleotide region (UOR), wherein a priming oligonucleotide produced in a reaction is capable of hybridizing to the ECT oligonucleotide and being extended across the universal oligonucleotide region as a template. In certain preferred embodiments, the ECT oligonucleotide comprises a snaking region configured to form a cleavage structure when said priming oligonucleotide has been extended on the ECT. In preferred embodiments, the cleavage structure is an invasive cleavage structure cleavable by a 5' nuclease.

In some embodiments, the composition is a reaction mixture. In some

embodiments, the composition is a kit (e.g., containing one or more containers, each housing one or more of the components). In some embodiments, a system of the invention comprises the composition and one or more additional components such as sample purification or processing reagents or equipment, detection equipment, control software, and data analysis systems.

In some embodiments, the composition comprises: a) a target nucleic acid; b) a primer; c) a polymerase; d) a FEN-1 endonuclease; and e) an extension/cleavage template (ECT) oligonucleotide comprising a snaking region, a portion complementary to a defined composition region (DCR), and a universal oligonucleotide region (UOR), wherein a priming oligonucleotide produced in a process comprising extending the primer is capable of hybridizing to the ECT oligonucleotide and being extended by the polymerase using the universal oligonucleotide region as a template, and wherein said snaking region forms an overlapping invasive cleavage structure after said priming oligonucleotide is extended on said ECT oligonucleotide, said invasive cleavage structure cleavable by said FEN-1. The present invention also provides methods of analyzing a target nucleic acid comprising: providing such a composition, forming a cleavage structure between an extended priming oligonucleotide and said ECT oligonucleotide, cleaving the cleavage structure with the FEN-1 endonuc lease to generate a cleavage product, and detecting the cleavage product.

DESCRIPTION OF THE DRAWINGS

Fig. 1A provides a diagram of an INVADER invasive cleavage assay. Panel a shows an overlapping invasive cleavage structure that is recognized and cleaved by a 5' nuclease such as an archaeal FEN-1 endonuclease, releasing a flap from the probe oligonucleotide. The released 5' flap is configured to form a secondary invasive cleavage structure with a hairpin-shaped FRET cassette. Cleavage of the FRET cassette separates a fluorophore (Fl) from a quencher moiety (Q), increasing detectable fluorescence from the fluorophore. Panel b illustrates the specificity of the reaction. When a probe directed to a mutant DNA hybridizes to a WT target, a mismatch with the target prevents formation of an overlap and the primary reaction structure is not cleaved. Without cleavage in the primary reaction, the FRET cassette is not cleaved in the secondary reaction and no signal develops. Use of differently labeled FRET cassettes (Fl and F2) allows detection of multiple different targets in a single reaction mixture.

Fig. IB provides a schematic diagram of one embodiment for creating a priming oligonucleotide having a defined composition region (DCR) and a distinct 3' end (D3E) region.

Fig. 2 shows a schematic diagram of one embodiment using temperature to displace a primer extension product. Fig. 3 shows a schematic diagram of one embodiment using strand-displacement to displace a primer extension product.

Fig. 4 shows a schematic diagram of one embodiment using duplex breathing and invasion by an oligonucleotide to displace a primer extension product.

Fig. 5 shows a diagram of an extension/cleavage template (ECT) oligonucleotide according to embodiments of the invention.

Fig. 6 shows a schematic diagram of an embodiment comprising use of an ECT oligonucleotide as a template to extend the Priming Oligonucleotide, wherein the 3' portion of the ECT oligonucleotide invades the newly-formed duplex (e.g., by a

"snaking" action) to form a cleavage structure. In some embodiments, cleavage of the extended Priming Oligonucleotide in the cleavage structure produces the original Priming Oligonucleotide such that the Priming oligonucleotide can be re -used in an additional extension reaction, e.g., on an ECT oligonucleotide.

Figs. 7 A, 7B, and 8 show embodiments of oligonucleotides configured for snaking strand invasion. The figures on the left show a structure in which the 3' end of the ECT oligonucleotide is not in a snaked configuration while the figures on the right show the same oligonucleotides in a snaked invasive cleavage structure. The snaked structures are designed to release the 5' arm from the probe (top strand) shown in lower case letters.

Fig. 9 shows results of cleavage reactions conducted as described in Example 1.

Fig. 10 shows embodiments of oligonucleotides configured for use in reactions in which a primer oligonucleotide is extended then cleaved in a snaking configuration, as described in Example 2.

Fig. 11 shows examples of different Priming oligonucleotides aligned to the "Complement of DCR" portions of the ECT oligonucleotides shown in Figure 10. The alignment shows mismatches between the Priming oligonucleotide DCR and the ECT oligonucleotide.

Fig. 12 shows results of extension/cleavage reactions conducted as described in Example 2.

Fig. 13 shows results of extension/cleavage reactions conducted as described in

Example 3. Fig. 14 shows results of extension/cleavage reactions conducted as described in Example 3.

Fig. 15 shows results of extension/cleavage reactions conducted as described in Example 3.

Fig. 16 shows results of extension/cleavage reactions conducted as described in

Example 3.

Fig. 17 shows a schematic diagram of an embodiment of the invention.

Fig. 18 shows a schematic diagram of an embodiment of the invention.

Fig. 19 provides a schematic diagram of an embodiment in which a reaction comprises a secondary reaction template (SRT) and a FRET-labeled oligonucleotide for detection of the priming oligonucleotide produced in a method according to the invention. In some embodiments, the SRT oligonucleotide comprises a first region complementary to the DCR of the priming oligonucleotide but not complementary to the 3' terminal nucleotide of the priming oligonucleotide, and a second region

complementary to the FRET oligonucleotide. In a reaction, a priming oligonucleotide and a FRET oligonucleotide anneal to the SRT to form a cleavage structure. In preferred embodiments, incubation the reaction under conditions in which the FRET

oligonucleotide cycles on and off the SRT (e.g., close to the Tm of the FRET

oligonucleotide/SRT duplex) allows cleavage of multiple FRET oligonucleotides per Priming Oligonucleotide/SRT complex.

DEFINITIONS

To facilitate an understanding of the present invention, a number of terms and phrases are defined below:

As used herein, the term "dynamic range" refers to the quantitative range of usefulness in a detection assay (e.g., a nucleic acid detection assay). For example, the dynamic range of a viral detection assay is the range between the smallest number of viral particles (e.g. , copy number) and the largest number of viral particles that the assay can distinguish between. As used herein, the terms "subject" and "patient" refer to any organisms including plants, microorganisms and animals (e.g., mammals such as dogs, cats, livestock, and humans).

The term "primer" refers to an oligonucleotide that is capable of acting as a point of initiation of synthesis when placed under conditions in which primer extension is initiated. An oligonucleotide "primer" may occur naturally, as in a purified restriction digest or may be produced synthetically. In some embodiments, an oligonucleotide primer is used with a template nucleic acid, extension of the primer is template dependent, such that a complement of the template is formed.

The term "priming oligonucleotide" as used herein refers to an oligonucleotide species that is created in the reaction (e.g., detection assay) in which it operates, generally by action of one or more nucleic acid modification enzymes (e.g., DNA polymerase, ligase, 5' nuclease). The priming oligonucleotide has a distinctive 3' end (D3E ) produced in the reaction, e.g., by cleavage of an extended primer, the D3E being capable of acting as a point of initiation of synthesis, e.g., on an ECT oligonucleotide or a secondary reaction template (SRT).

"Polymerase" means an enzyme capable of catalyzing template dependent oligonucleotide extension by conjugating extension nucleotides to an oligonucleotide or amplicon. In isothermal amplification processes, the polymerase generally promotes strand displacement, which refers to the ability of a polymerase to displace downstream DNA encountered during primer extension. DNA polymerases having strand

displacement activity include those of phi29 DNA polymerase, DNA polymerase I, Klenow fragment, Klenow fragment (3'→5' exo^"), DNA polymerases isolated or derived from thermophilic organisms, e.g., VENT® DNA Polymerase, 9°Nm DNA Polymerase, Therminator DNA Polymerase, Bacillus stearothermophilus (Bst) DNA polymerase

(U.S. Pat. Nos. 5,874,282; 6,100,078, and 6,066,483, Riggs et al), and the large fragment of Moloney murine leukemia virus (MMLV) reverse transcriptase (RT). In preferred embodiments, a Bst DNA polymerase may be modified to reduce, inhibit, inactivate or remove its 5' exonuclease activity (i.e., 5'-exo-minus polymerase). A polymerase may have reverse transcriptase (RT) activity that catalyzes extension of a DNA complement from an RNA template (i.e., RNA directed DNA polymerase), such as in MMLV RT and avian myeloblastosis virus (AMV) RT enzymes. RT activity may be provided in a fragment of a native polymerase. Preferred polymerases include those that tolerate modified oligonucleotides and/or modified extension nucleotides when catalyzing oligonucleotide extension.

A nucleic acid polymerase used in the isothermal amplification methods is an agent, generally an enzyme that incorporates RNA or DNA nucleotides or both, into a nucleic acid polymer in a template-dependent manner, usually in a 5' to 3' direction beginning at the 3' end of a primer. Examples of nucleic acid polymerases include DNA- directed DNA polymerases, RNA-directed DNA polymerases, and RNA-directed RNA polymerases. Preferred embodiments use a polymerase enzyme isolated from a thermophilic organism, e.g., Bst DNA polymerase or a modified version of a naturally occurring thermophilic polymerase enzyme.

Typically, during nucleic acid amplification, a nucleic acid polymerase adds nucleotides to the 3' end of a primer using the target nucleic acid strand as a template, thereby synthesizing a strand that includes a sequence partially or completely

complementary to a region of the target nucleic acid. In some reactions, the two strands of a resulting double-stranded nucleic acid are separated chemically or physically to allow amplification to proceed. Alternatively, a newly synthesized strand may be made available for binding to a primer by other means, e.g., use of strand displacement or a nucleolytic enzyme to digest part or all of a strand (e.g., the template strand), to allow cycle(s) of synthesis to produce many strands containing the target sequence or its complementary sequence.

"Extension nucleotides" refer to any nucleotide capable of being incorporated into an extension product during amplification, i.e., DNA, RNA, or a derivative if DNA or RNA, which may include a label.

"Osmolyte" means a molecule that contributes to the osmotic strength of an amplification system, which is added to some preferred embodiments to preferably enhance isothermal amplification. For example, osmolytes include but are not limited to betaine and/or trimethylamine N-oxide (TMAO). One or more osmolytes may be included, preferably at a concentration that mimics physiological concentrations, e.g., about 0.25M TMAO or about 1M betaine. Although not wishing to be bound to a particular theory or mechanism, an osmolyte in a reaction may interact with a polymerase to facilitate strand "breathing" which may not result in strand dissociation. Osmolytes that enhance assays such as isothermal detection assays may be identified by routine testing that compares results of assays that test different osmolytes compared to a control reaction that does not include the osmolyte, and selecting an osmolyte that enhances the reaction, e.g. increases signal in the reaction. The effects of osmolytes on isothermal amplification reactions in particular are described, e.g., in US Pat. Publication

2007/0054301, which is incorporated herein by reference in its entirety for all purposes.

Some embodiments do not include an osmolyte in the isothermal amplification reaction and, instead, a 3' end of nucleic acid strand, e.g., of a breathing end of a duplex acts as a primer to invade or "snake" into a double-stranded nucleic acid having a complementary breathing end, such that the strand having the 5' breathing end is displaced upon extension of the invading 3' end. In some embodiments a primer hybridizes to the displaced strand and primes synthesis of a new complementary strand. Related embodiments include an osmolyte in the reaction.

As used herein the terms "snake" and "snaking" refer to a configuration in which an end of one strand in a nucleic acid duplex invades the duplex, i.e., disrupts the duplex between the snaking strand and the strand to which it is base-paired. For example, in a complex in which a member of a duplex has an unpaired 3' portion or arm, the 3' end may wrap around and hybridize to an internal site on the same member strand, displacing the complementary strand from that region.

The term "cleavage structure" as used herein, refers to a structure that is formed by the interaction of at least one probe oligonucleotide and a target nucleic acid, forming a structure comprising a duplex, the resulting structure being cleavable by a cleavage means, including but not limited to an enzyme. The cleavage structure is a substrate for specific cleavage by the cleavage agent, in contrast to a nucleic acid molecule that is a substrate for non-specific cleavage by agents such as phosphodiesterases, which cleave nucleic acid molecules without regard to secondary structure (i.e., no formation of a duplexed structure is required).

The term "invasive cleavage structure" as used herein refers to a cleavage structure comprising i) a target nucleic acid, ii) an upstream nucleic acid (e.g. , an INVADER oligonucleotide), and iii) a downstream nucleic acid (e.g., a probe), where the upstream and downstream nucleic acids anneal to contiguous regions of the target nucleic acid, and where an overlap forms between the a 3' portion of the upstream nucleic acid and duplex formed between the downstream nucleic acid and the target nucleic acid. An overlap occurs where one or more bases from the upstream and downstream nucleic acids occupy the same position with respect to a target nucleic acid base, whether or not the overlapping base(s) of the upstream nucleic acid are complementary with the target nucleic acid, and whether or not those bases are natural bases or non-natural bases. In some embodiments, the 3' portion of the upstream nucleic acid that overlaps with the downstream duplex is a non-base chemical moiety such as an aromatic ring structure, e.g., as disclosed, for example, in U.S. Patent No. 6,090,543, incorporated herein by reference in its entirety. In some embodiments, one or more of the nucleic acids may be attached to each other, e.g., through a covalent linkage such as nucleic acid stem-loop, or through a non-nucleic acid chemical linkage (e.g., a multi-carbon chain).

The term "cleavage means" or "cleavage agent" as used herein refers to any means that is capable of cleaving a cleavage structure, including but not limited to enzymes. "Structure-specific nucleases" or "structure-specific enzymes" are enzymes that recognize specific secondary structures in a nucleic molecule and cleave these structures. The cleavage agent of the invention cleaves a nucleic acid molecule in response to the presence of a cleavage structure; it is not necessary that the cleavage agent cleave the cleavage structure at any particular location within the cleavage structure.

The cleavage agent may include nuclease activity provided from a variety of sources including the CLEAVASE enzymes from Hologic, Inc. (Marlborough, MA), the FEN-1 endonucleases (including RAD2 and XPG proteins, and natural or modified FEN- 1 enzymes or chimerical enzymes comprising at least a portion of one or more FEN-1 enzymes), and enzymes comprising a 5' nuclease activity, such as eubacterial PolA polymerases including but not limited to Taq DNA polymerase, Tth DNA polymerase and E. coli DNA polymerase I. The cleavage agent may also include modified DNA polymerases having 5' nuclease activity but lacking synthetic activity. Examples of cleavage agents suitable for use in the method and kits of the present invention are provided in U.S. Patent Nos. 5,614,402; 5,795,763; 5,843,669; 7,122,364, 7,150,982, and PCT Appln. Nos WO 98/23774; WO 02/070755A2; and WO0190337A2, each of which is herein incorporated by reference it its entirety. In certain preferred embodiments, the cleavage enzyme comprises an archaeal FEN-1, e.g., from Archaeoglobus fulgidus (AfuFEN), Archaeoglobus veneficus (AveFEN), Pyrococcus furiosus (PfuFEN),

Methanococcus jannaschii (MjaFEN), and Methanothermobacter thermoautotrophicum (MthFEN). See, e.g., Kaiser et al, J. Biol. Chem. Jul 23;274(30):21387-94 (1999) and WO 02/070755, each of which is incorporated herein by reference in its entirety. In certain preferred embodiments, the FEN-1 is from an Archaeoglobus species.

The term "thermostable" when used in reference to an enzyme, such as a 5' nuclease, indicates that the enzyme is functional or active (i.e., can perform catalysis) at an elevated temperature, i.e., at about 55°C or higher. In some embodiments the enzyme is functional or active at an elevated temperature of 65°C or higher (e.g., 75°C, 85°C, 95°C, etc.).

The term "cleavage products" as used herein, refers to products generated by the reaction of a cleavage agent with a cleavage structure (i.e., the treatment of a cleavage structure with a cleavage agent).

As used herein, the term "specifically hybridizes" means that under given hybridization conditions a probe or primer detectably hybridizes to substantially only the target sequence in a sample comprising the target sequence (i.e., there is little or no detectable hybridization to non-target sequences). In an amplification method that comprises cycles of denaturation and annealing of nucleic acid, e.g., targets and primers or probes, given hybridization conditions include the conditions for the annealing step in the amplification method, i.e., an annealing temperature selected on the basis of predicted T_m, and salt conditions suitable for the polymerase enzyme of choice.

The term "amplified" as used herein refers to an increase in the abundance of molecule, moiety or effect. A target nucleic acid may be amplified, e.g., by in vitro replication such as by PCR. A signal, e.g. , a detectable event or product that indicates the presence of a target nucleic acid

As used herein, the term "amplification method" as used in reference to nucleic acid amplification means a process of specifically amplifying the abundance of a nucleic acid of interest. Some amplification methods (e.g. , polymerase chain reaction, or PCR) comprise iterative cycles of thermal denaturation, oligonucleotide primer annealing to template molecules, and nucleic acid polymerase extension of the annealed primers. Conditions and times necessary for each of these steps are well known in the art. Some amplification methods are conducted under "isothermal" conditions. As used herein, "isothermal" means conducting a reaction at substantially constant temperature, i.e., without varying the reaction temperature in which a nucleic acid polymerization reaction occurs. Isothermal temperatures for isothermal amplification reactions are generally below the melting temperature (T_m; the temperature at which half of the potentially double-stranded molecules in a mixture are in a single-stranded, denatured state) of the predominant reaction product. In some configurations, an isothermal reaction may be conducted at a relatively high temperature, e.g., at 55°C-65°C, or higher, while in some embodiments, the assay is configured to run at lower temperatures (e.g., 25°C, 30°C, 37°C, 45°C, 50°C, etc.) Configuration for detection at lower temperatures may comprise use of nucleic acids having shorter duplexed regions (e.g., shorter probes or primer) use of enzymes having activity at lower temperatures, etc. Conversely, high temperature reactions may be favored by the use of longer nucleic acids (e.g., longer primers, probes, etc.) and the use of thermostable enzymes. Although the polymerization and cleavage reactions may occur in isothermal conditions, an isothermal process may optionally include a pre-amplification heat denaturation step to generate a single-stranded target nucleic acid to be used in the isothermal amplifying step.

Accumulation of the products of amplification may be exponential or linear. Some amplification methods ("target amplification" methods) amplify the abundance of a target sequence, e.g., by copying it many times (e.g., PCR, NASBA, TMA, strand displacement amplification, ligase chain reaction, LAMP, ICAN, RPA, SPIA, HAD,

INVADER Plus assay, Q-Invader assay, etc.), while some amplification methods amplify the abundance of a nucleic acid species that may or may not contain the target sequence, but the amplification of which indicates the presence of a particular target sequence in the reaction (e.g., INVADER assay, rolling circle amplification, RAM amplification). The latter methods are sometimes referred to as "signal amplification" methods. Some signal amplification methods may increase the abundance of a species of nucleic acid by converting a starting nucleic acid, e.g., by cleaving the starting nucleic acid to form cleavage products, or by extending it by, e.g., polymerization or ligation. A target amplification method may be applied to a signal molecule (e.g. , PCR may be used to produce more copies of the product of a ligation, cleavage, or non-target copying reaction), or vice versa.

As used herein, the terms "polymerase chain reaction" and "PCR" refer to an enzymatic reaction in which a segment of DNA is replicated from a target nucleic acid in vitro. The reaction generally involves extension of a primer on each strand of a target nucleic acid with a template dependent DNA polymerase to produce a complementary copy of a portion of that strand. The chain reaction comprises iterative cycles of denaturation of the DNA strands, e.g., by heating, followed by cooling to allow primer annealing and extension, resulting in an exponential accumulation of copies of the region of the target nucleic acid that is flanked by and that includes the primer binding sites. When an RNA target nucleic acid is amplified by PCR, it is generally first reverse transcribed to produce a DNA copy strand.

As used herein, the term "annealing" refers to conditions that permit

oligonucleotides, e.g., primers or probes, to hybridize to template nucleic acid strands. Conditions for primer annealing vary with the length and sequence of the primer and are generally based upon the T_m that is determined or calculated for the primer. For example, an annealing step in an amplification method that involves thermocycling involves reducing the temperature after a heat denaturation step to a temperature based on the T_m of the primer sequence, for a time sufficient to permit such annealing.

As used herein, the term "amplifiable nucleic acid" as used in reference to nucleic acids that may be amplified by any amplification method. It is contemplated that "amplifiable nucleic acid" will usually comprise "sample template."

The term "real time" as used herein in reference to detection of nucleic acid amplification or signal amplification refers to the detection or measurement of the accumulation of products or signal in the reaction while the reaction is in progress, e.g., during incubation or thermal cycling. Such detection or measurement may occur continuously, or it may occur at a plurality of discrete points during the progress of the amplification reaction, or it may be a combination. For example, in a polymerase chain reaction, detection (e.g. , of fluorescence) may occur continuously during all or part of thermal cycling, or it may occur transiently, at one or more points during one or more cycles. In some embodiments, real time detection of PCR is accomplished by

determining a level of fluorescence at the same point (e.g., a time point in the cycle, or temperature step in the cycle) in each of a plurality of cycles, or in every cycle. Real time detection of amplification may also be referred to as detection "during" the amplification reaction.

As used herein, the terms "reverse transcription" and "reverse transcribe" refer to the use of a template-dependent polymerase to produce a DNA strand complementary to an RNA template.

As used herein, the term "abundance of nucleic acid" refers to the amount of a particular target nucleic acid sequence present in a sample or aliquot. The amount is generally referred to in terms of mass (e.g., μgs), mass per unit of volume (e.g., μgs per μΐ); copy number (e.g., 1000 copies, 1 attomole), or copy number per unit of volume (e.g., 1000 copies per ml, 1 attomole per μΐ). Abundance of a nucleic acid can also be expressed as an amount relative to the amount of a standard of known concentration or copy number. Measurement of abundance of a nucleic acid may be on any basis understood by those of skill in the art as being a suitable quantitative representation of nucleic acid abundance, including physical density or the sample, optical density, refractive property, staining properties, or on the basis of the intensity of a detectable label, e.g. a fluorescent label.

The term "amplicon" or "amplified product" refers to a segment of nucleic acid, generally DNA, generated by an amplification process such as the PCR process. The terms are also used in reference to RNA segments produced by amplification methods that employ RNA polymerases, such as NASBA, TMA, etc.

The term "amplification plot" as used in reference to a thermal cycling

amplification reaction refers to the plot of signal that is indicative of amplification, e.g., fluorescence signal, versus cycle number. When used in reference to a non-thermal cycling amplification method, an amplification plot generally refers to a plot of the accumulation of signal as a function of time. The term "baseline" as used in reference to an amplification plot refers to the detected signal coming from assembled amplification reactions at prior to incubation or, in the case of PCR, in the initial cycles, in which there is little change in signal.

The term "Ct" or "threshold cycle" as used herein in reference to real time detection during an amplification reaction that is thermal cycled refers to the fractional cycle number at which the detected signal (e.g. , fluorescence) passes the fixed threshold.

The term "no template control" and "no target control" (or "NTC") as used herein in reference to a control reaction refers to a reaction or sample that does not contain template or target nucleic acid. It is used to verify amplification quality.

The term "passive reference" as used in reference to a detection reaction refers to a reference material, such as a dye, that provides an internal reference to which a reporter signal (e.g., another dye) can be normalized during data analysis. Normalization is generally necessary to correct for fluctuations caused by changes in concentration or volume.

"Rn" or "normalized reporter" refers to the fluorescence emission intensity of the reporter dye divided by the fluorescence emission intensity of the passive reference dye.

"Rn+" refers to the Rn value of a reaction containing all components, including the template or target.

Rn- refers to the Rn value of an un-reacted sample. The Rn- value can be obtained from the early cycles of a real time reaction, e.g., a real time PCR run (those cycles prior to a detectable increase in fluorescence), or from a reaction that does not contain any template.

"ARn" or "delta Rn" refers to the magnitude of the signal generated by the given set of amplification conditions, e.g., PCR conditions. The ARn value is determined by the following formula: (Rn+) - (Rn-) Standard A sample of known concentration used to construct a standard curve. By running standards of varying concentrations, one creates a standard curve from which one can extrapolate the quantity of an unknown sample.

The term "threshold" as used in reference to real time detection of an

amplification reaction refers to the average standard deviation of Rn for the early PCR cycles, multiplied by an adjustable factor. The threshold should be set in the region associated with an exponential growth of PCR product. The term "unknown" as used in reference to a quantitative assay refers to a sample containing an unknown quantity of template, generally a sample whose quantity one wants to determine, e.g., by performance of a quantitative assay such as a real time PCR and/or INVADER assay reaction.

As used herein, the term "sample template" refers to nucleic acid originating from a sample that is analyzed for the presence of "target." In contrast, "background template" is used in reference to nucleic acid other than sample template that may or may not be present in a sample. The presence of background template is most often inadvertent. It may be the result of carryover, or it may be due to the presence of nucleic acid contaminants sought to be purified away from the sample. For example, nucleic acids from organisms other than those to be detected may be present as background in a test sample.

The terms "analyte-specific region" or "ASR" and "analyte-specific portion" as used in reference to an oligonucleotide, such as a primer, a probe oligonucleotide, an ECT oligonucleotide, or an INVADER oligonucleotide, are used interchangeably and refer to a region/portion of an oligonucleotide selected to specifically hybridize to a particular nucleic acid sequence, e.g., in a target nucleic acid or set of target nucleic acids. In some embodiments, an analyte-specific region of an oligonucleotide may be completely complementary to the segment of a target nucleic acid to which it hybridizes, while in other embodiments, an analyte-specific region may comprise one or more mismatches to the segment of a target nucleic acid to which it hybridizes. In yet other embodiments, an analyte-specific region may comprise one or more base analogs, e.g., compounds that have altered hydrogen bonding, or that do not hydrogen bond, to the bases in the target strand. In some embodiments, the entire sequence of an

oligonucleotide is an analyte-specific region, while in other embodiments an

oligonucleotide comprises an analyte-specific region and one or more regions not complementary the target sequence (e.g., non-complementary flap regions).

The term "substantially single-stranded" when used in reference to a nucleic acid substrate means that the substrate molecule exists primarily as a single strand of nucleic acid in contrast to a double-stranded substrate which exists as two strands of nucleic acid which are held together by inter-strand base pairing interactions. The term "liberating" as used herein refers to the release of a nucleic acid fragment from a larger nucleic acid fragment, such as an oligonucleotide, by the action of, for example, a 5' nuclease such that the released fragment is no longer covalently attached to the remainder of the oligonucleotide.

The term "microorganism" as used herein means an organism too small to be observed with the unaided eye and includes, but is not limited to bacteria, virus, protozoans, fungi, and ciliates.

The term "microbial gene sequences" refers to gene sequences derived from a microorganism.

The term "bacteria" refers to any bacterial species.

The terms "archaea," "archaeal species," "archaean" and "archaebacteria" are used interchangeably refer to any organisms classified as a member of the Archaea domain or kingdom of life.

The term "virus" refers to obligate, ultramicroscopic, intracellular parasites incapable of autonomous replication (i.e., replication requires the use of the host cell's machinery).

The term "multi-drug resistant" or multiple-drug resistant" refers to a

microorganism that is resistant to more than one of the antibiotics or antimicrobial agents used in the treatment of said microorganism.

The term "source of target nucleic acid" refers to any sample that contains nucleic acids (R A or DNA). Particularly preferred sources of target nucleic acids are biological samples including, but not limited to blood, saliva, cerebral spinal fluid, pleural fluid, milk, lymph, sputum and semen.

A sample "suspected of containing" a first and a second target nucleic acid may contain either, both or neither target nucleic acid molecule.

The term "reactant" is used herein in its broadest sense. The reactant can comprise, for example, an enzymatic reactant, a chemical reactant or light (e.g. , ultraviolet light, particularly short wavelength ultraviolet light is known to break oligonucleotide chains). Any agent capable of reacting with an oligonucleotide to either shorten (i.e., cleave) or elongate the oligonucleotide is encompassed within the term "reactant." As used herein the term "portion" when in reference to a protein (as in "a portion of a given protein") refers to a fragment of that protein or a sequence of amino acids that is less than the complete chain of amino acids of the polypeptide. Similarly, when in reference to a nucleic acid (as in "a portion of a given nucleic acid or oligonucleotide"), the term refers to a fragment of a nucleic acid, or it refers to a sequence of nucleotides that is less than the complete chain of nucleotides of the nucleic acid or oligonucleotide. A portion may range in size from 1 amino acid or nucleotide residues, to the entire amino acid or nucleotide sequence.

The term "duplex" refers to the state of nucleic acids in which the base portions of the nucleotides on one strand are bound through hydrogen bonding the their

complementary bases arrayed on a second strand. The condition of being in a duplex form reflects on the state of the bases of a nucleic acid. By virtue of base pairing, the strands of nucleic acid also generally assume the tertiary structure of a double helix, having a major and a minor groove. The assumption of the helical form is implicit in the act of becoming duplexed.

The term "template" refers to a strand of nucleic acid on which a complementary copy is built from nucleoside triphosphates through the activity of a template-dependent nucleic acid polymerase. Within a duplex the template strand is, by convention, depicted and described as the "bottom" strand. Similarly, the non-template strand is often depicted and described as the "top" strand.

As used herein, the term "sample" is used in its broadest sense. For example, in some embodiments, it is meant to include a specimen or culture (e.g., microbiological culture), whereas in other embodiments, it is meant to include both biological and environmental samples (e.g., suspected of comprising a target sequence, gene or template). In some embodiments, a sample may include a specimen of synthetic origin. Samples may be unpurifed or may be partially or completely purified or otherwise processed.

The present invention is not limited by the type of biological sample used or analyzed. The present invention is useful with a variety of biological samples including, but are not limited to, tissue (e.g., organ (e.g., heart, liver, brain, lung, stomach, intestine, spleen, kidney, pancreas, and reproductive (e.g., ovaries) organs), glandular, skin, and muscle tissue), cell (e.g., blood cell (e.g., lymphocyte or erythrocyte), muscle cell, tumor cell, and skin cell), gas, bodily fluid (e.g., blood or portion thereof, serum, plasma, urine, semen, saliva, etc), or solid (e.g., stool) samples obtained from a human (e.g., adult, infant, or embryo) or animal (e.g., cattle, poultry, mouse, rat, dog, pig, cat, horse, and the like). In some embodiments, biological samples may be solid food and/or feed products and/or ingredients such as dairy items, vegetables, meat and meat by-products, and waste. Biological samples may be obtained from all of the various families of domestic animals, as well as feral or wild animals, including, but not limited to, such animals as ungulates, bear, fish, lagomorphs, rodents, etc.

Biological samples also include biopsies and tissue sections (e.g., biopsy or section of tumor, growth, rash, infection, or paraffin-embedded sections), medical or hospital samples (e.g., including, but not limited to, blood samples, saliva, buccal swab, cerebrospinal fluid, pleural fluid, milk, colostrum, lymph, sputum, vomitus, bile, semen, oocytes, cervical cells, amniotic fluid, urine, stool, hair and sweat), laboratory samples (e.g., subcellular fractions), and forensic samples (e.g., blood or tissue (e.g., spatter or residue), hair and skin cells containing nucleic acids), and archeological samples (e.g., fossilized organisms, tissue, or cells).

Environmental samples include, but are not limited to, environmental material such as surface matter, soil, water (e.g., freshwater or seawater), algae, lichens, geological samples, air containing materials containing nucleic acids, crystals, and industrial samples, as well as samples obtained from food and dairy processing instruments, apparatus, equipment, utensils, disposable and non-disposable items.

Other types of biological samples include bacteria (e.g., Actinobacteria (e.g., Actinomyces, Arthrobacter, Corynebacterium (e.g., C. diphtheriae)), Mycobacterium (e.g., M. tuberculosis and leprae), Propionibacterium (e.g., P. acnes), Streptomyces, hlamydiae (e.g., C. trachomatis and C. pneumoniae), Cyanobacteria, Deinococcus (e.g., Thermus (e.g., T. aquaticus ), Firmicutes (e.g., Bacilli (e.g., B. anthracis, B. cereus, B. thuringiensis, and B. subtilis)), Listeria (e.g., L. monocytogenes), Staphylococcus (e.g., S. aureus, S. epidermidis, and S. haemolyticus), Fusobacteria, Proteobacteria (e.g., Rickettsiales, Sphingomonadales, Bordtella (e.g., B. pertussis), Neisserisales (e.g., N. gonorrhoeae and N. meningitidis), Enterobacteriales (e.g., Escherichia (e.g., E. coli), Klebsiella, Plesiomonas, Proteus, Salmonella, Shigella, and Yersinia), Legionellales, Pasteur ellales (e.g., Haemophilus influenzae), Pseudomonas, Vibrio (e.g., V. cholerae and V. vulnificus), Campylobacter ales (e.g., Campylobacteria (e.g., C. jejuni), and Helicobacter (e.g., H. pylori)), and Spirochaetes (e.g., Leptospira, B. bergdorferi, and T. pallidum)); Archaea (e.g., Halobacteria and Methanobacteria); Eucarya (e.g., Animalia (e.g., Annelidia, Arthropoda (e.g., Chelicerata, Myriapoda, Insecta, and Crustacea), Mollusca, Nematoda,( e.g., C. elegans, and T. spiralis) and Chordata (e.g.,

Actinopterygii, Amphibia, Aves, Chondrichthyes, Reptilia, and Mammalia (e.g., Primates, Rodentia, Lagomorpha, and Carnivora)))); Fungi (e.g., Dermatophytes, Fusarium, Penicillum, and Saccharomyces); Plantae (e.g., Magnoliophyta (e.g., Magnoliopsida and Liliopsida)), and Protista (e.g., Apicomplexa (e.g., Cryptosporidium, Plasmodium (e.g., P. falciparum, and Toxoplasma), and Metamonada (e.g., G. lambia))); and Viruses (e.g., dsDNA viruses (e.g., Bacteriophage, Adenoviridae, Herpesviridiae, Papillomaviridae, Polyomaviridae, and Poxviridae), ssDNA virues (e.g., Parvoviridae), dsR A viruses (including Reoviridae), (+)ssRNA viruses (e.g., Coronaviridae, Astroviridae,

Bromoviridae, Comoviridae, Flaviviridae, Picornaviridae, and Togaviridae), (-) ssR A viruses (e.g., Bornaviridae, Filoviridae, Paramyxoviridae, Rhabdoviridae, Bunyaviridae, and Orthomyxovirdiae), ssR A-reverse transcribing viruses (e.g., Retroviridae), and dsDNA-reverse transcribing viruses (e.g., Hepadnaviridae and Caulomoviridae)).

Sample may be prepared by any desired or suitable method. In some

embodiments, nucleic acids are analyzed directly from bodily fluids or other samples using the methods described in U.S. Pat. Pub. Serial No. 20050186588, herein

incorporated by reference in its entirety.

The above described examples are not, however, to be construed as limiting the sample (e.g., suspected of comprising a target sequence, gene or template (e.g., the presence or absence of which can be determined using the compositions and methods of the present invention) types applicable to the present invention.

The term "nucleotide analog", "non-natural", or "non-naturally occurring" as used herein refers to nucleotides other than the natural nucleotides and bases. Such analogs and non-natural bases and nucleotides include modified natural nucleotides and non- naturally occurring nucleotides, including but not limited to analogs that have altered stacking interactions such as 7-deaza purines (i.e., 7-deaza-dATP and 7-deaza-dGTP); base analogs with alternative hydrogen bonding configurations (e.g., such as iso-C and iso-G and other non-standard base pairs described in U.S. Patent No. 6,001,983 to S. Benner, and the selectively binding base analogs described in U.S. Patent No. 5,912,340 to Igor V. Kutyavin, et al.); non-hydrogen bonding analogs (e.g., non-polar, aromatic nucleoside analogs such as 2,4-difluorotoluene, described by B.A. Schweitzer and E.T. Kool, J. Org. Chem., 1994, 59, 7238-7242, B.A. Schweitzer and E.T. Kool, J. Am. Chem. Soc, 1995, 1 17, 1863-1872); "universal" bases such as 5-nitroindole and 3-nitropyrrole; and universal purines and pyrimidines (such as "K" and "P" nucleotides, respectively; P. Kong, et al, Nucleic Acids Res., 1989, 17, 10373-10383, P. Kong et al, Nucleic Acids Res., 1992, 20, 5149-5152). Nucleotide analogs include modified forms of

deoxyribonucleotides as well as ribonucleotides. "Non-natural" and "non-naturally occurring" bases and nucleotides are specifically not limited to such bases as are never found in nature. Natural processes such as nucleic acid damage can give rise to "natural" occurrence of bases that are nonetheless not generally considered to be part of the set of "natural" nucleotides as defined herein. For example, iso-G can be found in oxidatively damaged DNA. Such non-natural bases and their behaviors in replication and other nucleic acid syntheses have been extensively studied in contexts such as DNA damage studies, although the compounds are sometimes described using different nomenclature. For example, the ribonucleoside comprising the isoguanosine base has been referred to in the literature variously as: iG; isoG; iso-G; isoguanosine; 2-hydroxyadenine; 2- oxoadenine; 2-hydroxy A; and 2-OH-A. The deoxyribonucleoside comprising the isoguanosine base has been referred to variously as: iG; isoG; iso dG; deoxyiso-G;

deoxyisoguanosine; 2-hydroxydeoxyadenosine; 2-hydroxy dA; and 2-OH-Ade.

Still other nucleotide analogs include modified forms of deoxyribonucleotides as well as ribonucleotides. Various oligonucleotides of the present invention (e.g., a primary probe or INVADER oligo) may contain nucleotide analogs.

The terms "nucleic acid sequence" and "nucleic acid molecule" as used herein refer to an oligonucleotide, nucleotide or polynucleotide, and fragments or portions thereof. The terms encompasses sequences that include analogs of DNA and RNA nucleotides, including those listed above, and also including, but not limited to, 4- acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5- (carboxyhydroxyl-methyl) uracil, 5-fluorouracil, 5-bromouracil, 5- carboxymethylaminomethyl-2-thiouracil, 5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudo-uracil, 1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine, 2-methyladenine, 2- methylguanine, 3-methyl-cytosine, 5-methylcytosine, N6-methyladenine, 7- methylguanine, 5-methylaminomethyluracil, 5-methoxy-amino-methyl-2-thiouracil, beta- D-mannosylqueosine, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio- N6-isopentenyladenine, uracil-5-oxyacetic acid methylester, uracil-5-oxy acetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2- thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxy acetic acid methylester, uracil-5- oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, 2,6-diaminopurine, and pyrazolo[3,4-d]pyrimidines such as guanine analogue 6 amino 1H- pyrazolo[3,4d]pyrimidin 4(5H) one (ppG or PPG, also Super G) and the adenine analogue 4 amino lH-pyrazolo[3,4d]pyrimidine (ppA or PPA). The xanthine analogue lH-pyrazolo[5,4d]pyrimidin 4(5H)-6(7H)-dione (ppX) can also be used. These base analogues, when present in an oligonucleotide, strengthen hybridization and improve mismatch discrimination. All tautomeric forms of naturally-occurring bases, modified bases and base analogues may be included in the oligonucleotide conjugates of the invention. Other modified bases useful in the present invention include 6-amino-3-prop- l-ynyl-5-hydropyrazolo[3,4-d]pyrimidine-4-one, PPPG; 6-amino-3 -(3 -hydroxyprop- 1- yny)l-5-hydropyrazolo[3,4-d]pyrimidine-4-one, HOPPPG; 6-amino-3-(3-aminoprop-l- ynyl)-5-hydropyrazolo[3,4-d]pyrimidine-4- -one, NH2PPPG; 4-amino-3-(prop-l- ynyl)pyrazolo [3 ,4-d]pyrimidine, PPP A; 4-amino-3 -(3 -hydroxyprop- 1 -ynyl)pyrazolo [3 ,4- d]pyrimidine, HOPPPA; 4-amino-3-(3-aminoprop-l-ynyl)pyrazolo[3,4-d]pyrimidine,

NH₂ PPP A; 3-prop-l-ynylpyrazolo[3,4-d]pyrimidine-4,6-diamino, (NH₂)₂ PPP A; 2-(4,6- diaminopyrazolo[3,4-d]pyrimidin-3-yl)ethyn-l-ol, (NH₂) ₂ PPPAOH; 3-(2- aminoethynyl)pyrazolo[3,4-d]pyrimidine-4,6-diamine, (NH₂) ₂ PPPANH₂; 5-prop-l-ynyl- l,3-dihydropyrimidine-2,4-dione, PU; 5-(3-hydroxyprop-l-ynyl)-l,3-dihydropyrimidine- 2,4-dione, HOPU; 6-amino-5-prop-l-ynyl-3-dihydropyrimidine-2-one, PC; 6-amino-5- (3-hydroxyprop-l-yny)-l,3-dihydropyrimidine-2-one, HOPC; and 6-amino-5-(3- aminoprop- 1 -yny)- 1 ,3 -dihydropyrimidine-2-one, NH₂PC ; 5 - [4-amino-3 -(3 -methoxyprop- l-ynyl)pyrazol[3,4-d]pyrimidinyl]-2-(hydroxym- ethyl)oxolan-3-ol, CH₃ OPPPA; 6- amino- 1 - [4-hydroxy-5 -(hydroxymethyl)oxolan-2-yl] -3 -(3 -methoxyprop- 1 -yny- 1)-5 - hydropyrazolo[3,4-d]pyrimidin-4-one, CH₃ OPPPG; 4,(4,6-Diamino-lH-pyrazolo[3,4- d]pyrimidin-3-yl)-but-3-yn-l-ol, Super A; 6-Amino-3-(4-hydroxy-but-l-ynyl)-l ,5- dihydro-pyrazolo[3,4-d]pyrimidin-4-o- ne; 5-(4-hydroxy-but-l-ynyl)-lH-pyrimidine-2,4- dione, Super T; 3-iodo-lH-pyrazolo[3,4-d]pyrimidine-4,6-diamine ((NH₂) ₂PPAI); 3- bromo-lH-pyrazolo[3,4-d]pyrimidine-4,6-diamine ((NH₂)₂ PPABr); 3-chloro-lH- pyrazolo[3,4-d]pyrimidine-4,6-diamine ((NH₂) ₂PPAC1); 3-Iodo-lH-pyrazolo[3,4- d]pyrimidin-4-ylamine (PPAI); 3-Bromo-lH-pyrazolo[3,4-d]pyrimidin-4-ylamine (PPABr); and 3-chloro-lH-pyrazolo[3,4-d]pyrimidin-4-ylamine (PPAC1).

In addition to the modified bases noted above, oligonucleotides of the invention can have a backbone of sugar or glycosidic moieties, preferably 2-deoxyribofuranosides wherein all internucleotide linkages are the naturally occurring phosphodiester linkages. In alternative embodiments however, the 2-deoxy-P-D-ribofuranose groups are replaced with other sugars, for example, β-D-ribofuranose. In addition, β-D-ribofuranose may be present wherein the 2-OH of the ribose moiety is alkylated with a Ci_₆ alkyl group (2-(0— Ci_6 alkyl) ribose) or with a C₂_6 alkenyl group (2-(0— C₂-₆ alkenyl) ribose), or is replaced by a fluoro group (2-fluororibose). Related oligomer-forming sugars useful in the present invention are those that are "locked", i.e. , contain a methylene bridge between C-4' and an oxygen atom at C-2'. Other sugar moieties compatible with hybridization of the oligonucleotide can also be used, and are known to those of skill in the art, including, but not limited to, α-D-arabinofuranosides, a-2'-deoxyribofuranosides or 2',3'-dideoxy-3'- aminoribofuranosides. Oligonucleotides containing a-D-arabinofuranosides can be prepared as described in U.S. Pat. No. 5, 177, 196. Oligonucleotides containing 2', 3'- dideoxy-3'-aminoribofuranosides are described in Chen et al. Nucleic Acids Res.

23 :2661-2668 (1995). Synthetic procedures for locked nucleic acids (Singh et al, Chem. Comm., 455-456 (1998); Wengel J., Acc. Chem. Res., 32:301-310 (1998)) and oligonucleotides containing 2'-halogen-2'-deoxyribofuranosides (Palissa et al, Z. Chem., 27:216 (1987)) have also been described. The phosphate backbone of the modified oligonucleotides described herein can also be modified so that the oligonucleotides contain phosphorothioate linkages and/or methylphosphonates and/or phosphoroamidates (Chen et al, Nucl. Acids Res., 23 :2662-2668 (1995)). Combinations of oligonucleotide linkages are also within the scope of the present invention. Still other backbone modifications are known to those of skill in the art.

In some embodiments, the modified bases described herein are incorporated into

PNA and DNA/PNA chimeras to balance T_ms and provide modified oligonucleotides having improved mismatch discrimination. Various modified forms of DNA and DNA analogues have been used in attempts to overcome some of the disadvantages of the use of DNA molecules as probes and primers. Among these are peptide nucleic acids (PNAs, also known as polyamide nucleic acids). Nielsen et al. Science 254: 1497-1500 (1991). PNAs contain heterocyclic base units, as found in DNA and RNA, that are linked by a polyamide backbone, instead of the sugar-phosphate backbone characteristic of DNA and RNA. PNAs are capable of hybridization to complementary DNA and RNA target sequences and, in fact, hybridize more strongly than a corresponding nucleic acid probe. The synthesis of PNA oligomers and reactive monomers used in the synthesis of PNA oligomers have been described in U.S. Pat. Nos. 5,539,082; 5,714,331 ; 5,773,571 ;

5,736,336 and 5,766,855. Alternate approaches to PNA and DNA/PNA chimera synthesis and monomers for PNA synthesis have been summarized. Uhlmann et al.

Angew. Chem. Int. Ed. 37:2796-2823 (1998). Accordingly, the use of any combination of normal bases, unsubstituted pyrazolo[3,4-d]pyrimidine bases (e.g., PPG and PPA), 3- substituted pyrazolo[3,4-d]pyrimidines, modified purine, modified pyrimidine, 5- substituted pyrimidines, universal bases, sugar modification, backbone modification or a minor groove binder to balance the T_m of a DNA, PNA or DNA/PNA chimera is in the scope of this invention. The synthetic methods necessary for the synthesis of modified base monomeric units required for nucleic acid, PNA and PNA/DNA chimeras synthesis are available in the art, see methods in this application and Uhlmann et al. Angew. Chem. Int. Ed. 37:2796-2823 (1998).

A nucleic acid sequence or molecule may be DNA or RNA, of either genomic or synthetic origin, that may be single or double stranded, and represent the sense or antisense strand. Thus, nucleic acid sequence may be dsDNA, ssDNA, mixed ssDNA, mixed dsDNA, dsDNA made into ssDNA (e.g., through melting, denaturing, helicases, etc.), A-, B-, or Z- DNA, triple-stranded DNA, R A, ssRNA, dsRNA, mixed ss and dsRNA, dsRNA made into ssRNA (e.g. , via melting, denaturing, helicases, etc.), messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), catalytic RNA, snRNA, microRNA, or protein nucleic acid (PNA).

The present invention is not limited by the type or source of nucleic acid (e.g., sequence or molecule (e.g. target sequence and/or oligonucleotide)) utilized. For example, the nucleic acid sequence may be amplified or created sequence (e.g., amplification or creation of nucleic acid sequence via synthesis (e.g. , polymerization (e.g. , primer extension (e.g. , RNA-DNA hybrid primer technology)) and reverse transcription (e.g., of RNA into DNA)) and/or amplification (e.g. , polymerase chain reaction (PCR), rolling circle amplification (RCA), nucleic acid sequence based amplification (NASBA), transcription mediated amplification (TMA), ligase chain reaction (LCR), cycling probe technology, Q-beta replicase, strand displacement amplification (SDA), branched-DNA signal amplification (bDNA), hybrid capture, and helicase dependent amplification).

The terms "nucleotide" and "base" are used interchangeably when used in reference to a nucleic acid sequence, unless indicated otherwise herein.

The term "oligonucleotide" as used herein is defined as a molecule comprising two or more nucleotides (e.g., deoxyribonucleotides or ribonucleotides), preferably at least 5 nucleotides, more preferably at least about 10-15 nucleotides and more preferably at least about 15 to 30 nucleotides, or longer (e.g. , oligonucleotides are typically less than 200 residues long (e.g., between 15 and 100 nucleotides), however, as used herein, the term is also intended to encompass longer polynucleotide chains). The exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide. Oligonucleotides are often referred to by their length. For example a 24 residue oligonucleotide is referred to as a "24-mer". Oligonucleotides can form secondary and tertiary structures by self-hybridizing or by hybridizing to other polynucleotides. Such structures can include, but are not limited to, duplexes, hairpins, cruciforms, bends, and triplexes. Oligonucleotides may be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, PCR, or a combination thereof. In some embodiments, oligonucleotides that form invasive cleavage structures are generated in a reaction (e.g., by extension of a primer in an enzymatic extension reaction).

Because mononucleotides are reacted to make oligonucleotides in a manner such that the 5' phosphate of one mononucleotide pentose ring is attached to the 3' oxygen of its neighbor in one direction via a phosphodiester linkage, an end of an oligonucleotide is referred to as the "5' end" if its 5' phosphate is not linked to the 3' oxygen of a

mononucleotide pentose ring and as the "3' end" if its 3' oxygen is not linked to a 5' phosphate of a subsequent mononucleotide pentose ring. As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide, also may be said to have 5' and 3' ends. A first region along a nucleic acid strand is said to be upstream of another region if the 3' end of the first region is before the 5' end of the second region when moving along a strand of nucleic acid in a 5' to 3' direction.

When two different, non-overlapping oligonucleotides anneal to different regions of the same linear complementary nucleic acid sequence, and the 3' end of one oligonucleotide points towards the 5' end of the other, the former may be called the

"upstream" oligonucleotide and the latter the "downstream" oligonucleotide. Similarly, when two overlapping oligonucleotides are hybridized to the same linear complementary nucleic acid sequence, with the first oligonucleotide positioned such that its 5' end is upstream of the 5' end of the second oligonucleotide, and the 3' end of the first oligonucleotide is upstream of the 3' end of the second oligonucleotide, the first oligonucleotide may be called the "upstream" oligonucleotide and the second

oligonucleotide may be called the "downstream" oligonucleotide.

As used herein, the terms "complementary" or "complementarity" are used in reference to polynucleotides (e.g., a sequence of two or more nucleotides (e.g., an oligonucleotide or a target nucleic acid)) related by the base-pairing rules. For example, the sequence "5'-A-G-T-3',^M is complementary to the sequence "3'-T-C-A-5\"

Complementarity may be "partial," in which only some of the nucleic acid bases are matched according to the base pairing rules. Or, there may be "complete" or "total" complementarity between the nucleic acid bases. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods that depend upon the association of two or more nucleic acid strands. Either term may also be used in reference to individual nucleotides, especially within the context of polynucleotides. For example, a particular nucleotide within an oligonucleotide may be noted for its complementarity, or lack thereof, to a nucleotide within another nucleic acid sequence (e.g., a target sequence), in contrast or comparison to the complementarity between the rest of the oligonucleotide and the nucleic acid sequence.

The complement of a nucleic acid sequence as used herein refers to an

oligonucleotide which, when aligned with the nucleic acid sequence such that the 5' end of one sequence is paired with the 3' end of the other, is in "antiparallel association." Nucleotide analogs, as discussed above, may be included in the nucleic acids of the present invention. Complementarity need not be perfect; stable duplexes may contain mismatched base pairs or unmatched bases. Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length of the oligonucleotide, base composition and sequence of the oligonucleotide, ionic strength and incidence of mismatched base pairs.

The term "homology" refers to a degree of complementarity. There may be partial homology or complete homology (i.e. , identity). A partially homologous sequence is one that is less than 100% identical to another sequence. A partially complementary sequence that is "substantially homologous" is a nucleic acid molecule that at least partially inhibits a completely complementary nucleic acid molecule from hybridizing to a target nucleic acid. The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (e.g. , Southern or Northern blot, solution hybridization and the like) under conditions of low stringency. A substantially homologous sequence or probe will compete for and inhibit the binding (e.g., the hybridization) of a completely homologous nucleic acid molecule to a target under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific binding is permitted (e.g., the low stringency conditions may be such that the binding of two sequences to one another be a specific (e.g., selective) interaction). The absence of non-specific binding may be tested by the use of a second target that is substantially non-complementary (e.g. , less than about 30% identity); in the absence of non-specific binding the probe will not hybridize to the second non-complementary target.

When used in reference to a double-stranded nucleic acid sequence such as a cDNA or genomic clone, the term "substantially homologous" refers to any probe that can hybridize to either or both strands of the double-stranded nucleic acid sequence under conditions of low stringency as described above.

When used in reference to a single-stranded nucleic acid sequence, the term "substantially homologous" refers to any probe that can hybridize (e.g., is complementary to) the single-stranded nucleic acid sequence under conditions of low stringency as described above.

The terms "target nucleic acid" and "target sequence," refers to a nucleic acid of to be detected or analyzed. Thus, the "target" is sought to be distinguished from other nucleic acids or nucleic acid sequences. For example, when used in reference to an amplification reaction, these terms may refer to the nucleic acid or portion of nucleic acid that will be amplified by the reaction, while when used in reference to a polymorphism, they may refer to the portion of an containing a suspected polymorphism. When used in reference to an invasive cleavage reaction, these terms refer to a nucleic acid molecule containing a sequence that has at least partial complementarity with at least a first nucleic acid molecule (e.g. probe oligonucleotide) and may also have at least partial

complementarity with a second nucleic acid molecule (e.g. INVADER oligonucleotide). Generally, the target nucleic acid (e.g. , present within, isolated from, enriched from, or amplified from or within a sample (e.g. , a biological or environmental sample)) is located within a target region and is identifiable via the successful formation of an invasive cleavage structure in combination with a first and second nucleic acid molecule (e.g., probe oligonucleotide and INVADER oligonucleotide) that is cleavable by a cleavage agent. Target nucleic acids from an organism are not limited to genomic DNA and RNA. Target nucleic acids from an organism may comprise any nucleic acid species, including but not limited to genomic DNAs and RNAs, messenger RNAs, structural RNAs, ribosomal and tRNAs, and small RNAs such as snRNAs, siRNAs and microRNAs (miRNAs). See, e.g., co-pending U.S. Patent Application Ser. No. 10/740,256, filed 12/18/03, which is incorporated herein by reference in its entirety. A "segment" is defined as a region of nucleic acid within the target sequence.

As used herein, the term "probe oligonucleotide," refers to an oligonucleotide that interacts with a target nucleic acid to form a detectable complex. In 5' nuclease cleavage assays such as the TAQMAN assay and the INVADER assay, the probe oligonucleotide hybridizes to the target nucleic acid and cleavage occurs within the probe

oligonucleotide. In some embodiments, the complex between a probe and target is detected while it exists, while in some embodiments, the formation of the complex may be detected when it no longer exits, e.g., by detection of an event {e.g., a cleavage event) that occurred as a result of formation of the probe/target complex.

The term "INVADER oligonucleotide" refers to an oligonucleotide that hybridizes to a target nucleic acid at a location near the region of hybridization between a probe and the target nucleic acid, wherein the INVADER oligonucleotide comprises a portion {e.g. , a chemical moiety, or nucleotide, whether complementary to that target or not) that overlaps with the region of hybridization between the probe and target. In some embodiments, the INVADER oligonucleotide contains sequences at its 3' end that are substantially the same as sequences located at the 5' end of a probe oligonucleotide.

The term "cassette," as used herein refers to an oligonucleotide or combination of oligonucleotides configured to generate a detectable signal in response to cleavage of a probe oligonucleotide. In preferred embodiments, the cassette hybridizes to a cleavage product from cleavage of the probe oligonucleotide to form a second invasive cleavage structure, such that the cassette can then be cleaved.

In some embodiments, the cassette is a single oligonucleotide comprising a hairpin portion (i.e., a region wherein one portion of the cassette oligonucleotide hybridizes to a second portion of the same oligonucleotide under reaction conditions, to form a duplex). In other embodiments, a cassette comprises at least two oligonucleotides comprising complementary portions that can form a duplex under reaction conditions. In preferred embodiments, the cassette comprises a label. In particularly preferred embodiments, the cassette comprises labeled moieties that produce a fluorescence resonance energy transfer (FRET) effect. As used herein, the term "universal" as used in reference to a nucleotide or nucleotide sequence refers to a base or sequence that is not specific to a particular target nucleic acid, e.g., that can be used in assays for the detection of any or all target nucleic acids. For example, an oligonucleotide may be constructed to comprise a portion that is "target specific", i.e., that has a sequence selected to hybridize with a specific target sequence, and portion that is selected to not hybridize to the target, e.g., that has a universal sequence that can be detected in a target-independent manner.

An oligonucleotide is said to be present in "excess" relative to another

oligonucleotide (or target nucleic acid sequence) if that oligonucleotide is present at a higher molar concentration than the other oligonucleotide (or target nucleic acid sequence). When an oligonucleotide such as a probe oligonucleotide is present in a cleavage reaction in excess relative to the concentration of the complementary target nucleic acid sequence, the reaction may be used to indicate the amount of the target nucleic acid present. Typically, when present in excess, the probe oligonucleotide will be present in at least a 100-fold molar excess; typically at least 1 pmole of each probe oligonucleotide would be used when the target nucleic acid sequence was present at about 10 fmoles or less.

As used herein, the term "gene" refers to a nucleic acid (e.g. , DNA) sequence that comprises coding sequences necessary for the production of a polypeptide, precursor, or R A (e.g. , rRNA, tR A). The polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or functional properties (e.g. , enzymatic activity, ligand binding, signal transduction, immunogenicity, etc.) of the full-length or fragment polypeptide are retained. The term also encompasses the coding region of a structural gene and the sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of about 1 kb or more on either end such that the gene corresponds to the length of the full-length mRNA. Sequences located 5' of the coding region and present on the mRNA are referred to as 5' non-translated sequences. Sequences located 3' or downstream of the coding region and present on the mRNA are referred to as 3' non-translated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene. A genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed "introns" or "intervening regions" or "intervening sequences." Introns are segments of a gene that are transcribed into nuclear RNA (e.g. , hnRNA); introns may contain regulatory elements (e.g., enhancers). Introns are removed or "spliced out" from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.

As used herein, the term "heterologous gene" refers to a gene that is not in its natural environment. For example, a heterologous gene includes a gene from one species introduced into another species (e.g., a viral or bacterial gene present within a human host (e.g., extrachromosomally or integrated into the host's DNA)). A heterologous gene also includes a gene native to an organism that has been altered in some way (e.g., mutated, added in multiple copies, linked to non-native regulatory sequences, etc). In some embodiments, a heterologous gene can be distinguished from endogenous genes in that the heterologous gene sequences are typically joined to DNA sequences that are not found naturally associated with the gene sequences in the chromosome or are associated with portions of the chromosome not found in nature (e.g., genes expressed in loci where the gene is not normally expressed).

As used herein, the term "gene expression" refers to the process of converting genetic information encoded in a gene into RNA (e.g. , mRNA, rRNA, tRNA, or snRNA) through "transcription" of the gene (e.g. , via the enzymatic action of an RNA

polymerase), and for protein encoding genes, into protein through "translation" of mRNA. Gene expression can be regulated at many stages in the process. "Up- regulation" or "activation" refers to regulation that increases the production of gene expression products (e.g., RNA or protein), while "down-regulation" or "repression" refers to regulation that decrease production. Molecules (e.g., transcription factors) that are involved in up-regulation or down-regulation are often called "activators" and "repressors," respectively.

In addition to containing introns, genomic forms of a gene may also include sequences located on both the 5' and 3' end of the sequences that are present on the RNA transcript. These sequences are referred to as "flanking" sequences or regions (e.g., these flanking sequences can be located 5 ' or 3' to the non-translated sequences present on the mR A transcript). The 5' flanking region may contain regulatory sequences such as promoters and enhancers that control or influence the transcription of the gene. The 3' flanking region may contain sequences that direct the termination of transcription, post-transcriptional cleavage and polyadenylation.

The term "wild-type" refers to a gene or gene product isolated from a naturally occurring source. A wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designed the "normal" or "wild-type" form of the gene. In contrast, the term "modified" or "mutant" refers to a gene or gene product that displays modifications in sequence and or functional properties (e.g., altered characteristics) when compared to the wild-type gene or gene product. It is noted that naturally occurring mutants can be isolated (e.g., identified by the fact that they have altered characteristics (e.g., altered nucleic acid sequences) when compared to the wild-type gene or gene product).

The term "isolated" when used in relation to a nucleic acid (e.g., "an isolated oligonucleotide" or "isolated polynucleotide" or "an isolated nucleic acid sequence") refers to a nucleic acid sequence that is separated from at least one component or contaminant with which it is ordinarily associated in its natural source. Thus, an isolated nucleic acid is present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated nucleic acids are nucleic acids such as DNA and RNA found in the state they exist in nature. For example, a given DNA sequence (e.g. , a gene) is found on the host cell chromosome in proximity to neighboring genes; RNA sequences, such as a specific mRNA sequence encoding a specific protein, are found in the cell as a mixture with numerous other mRNAs that encode a multitude of proteins. However, isolated nucleic acid encoding a given protein includes, by way of example, such nucleic acid in cells ordinarily expressing the given protein where the nucleic acid is in a chromosomal location different from that of natural cells, or is otherwise flanked by a different nucleic acid sequence than that found in nature. The isolated nucleic acid, oligonucleotide, or polynucleotide may be present in single-stranded or double-stranded form. When an isolated nucleic acid, oligonucleotide or polynucleotide is to be utilized to express a protein, the oligonucleotide or polynucleotide will contain at a minimum the sense or coding strand (e.g., the oligonucleotide or polynucleotide may be single - stranded), but may contain both the sense and anti-sense strands (e.g. , the oligonucleotide or polynucleotide may be double-stranded).

As used herein, the terms "purified" or "to purify" when used in reference to a sample (e.g. , a molecule (e.g. , a nucleic acid or amino acid sequence)) refers to removal (e.g. , isolation and/or separation) of the sample from its natural environment. The term "substantially purified" refers to a sample (e.g., molecule (e.g. a nucleic acid or amino acid sequence) that has been removed (e.g. , isolated and/or purified) from its natural environment and is at least 60% free, preferably 75% free, or most preferably 90% or more free from other components with which it is naturally associated. An "isolated polynucleotide" or "isolated oligonucleotide" may therefore be substantially purified if it is rendered free (e.g., 60%, 75% or more preferably 90%> or more) from other components with which it is naturally associated.

The present invention is not limited to any particular means of purification (e.g., to generate purified or substantially purified molecules (e.g. , nucleic acid sequences)). Indeed, a variety of purification techniques may be utilized including, but not limited to, centrifugation (e.g. , isopycnic, rate -zonal, gradient, and differential centrifugation), electrophoresis (e.g., gel and capillary electrophoresis), gel filtration, matrix capture, charge capture, mass capture, antibody capture, magnetic separation, flow cytometry, and sequence-specific hybridization array capture.

As used herein, the term "hybridization" is used in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (e.g., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementary between the nucleic acids, stringency of the conditions involved, the T_m of the formed hybrid, and the G:C ratio within the nucleic acids. A single molecule that contains pairing of complementary nucleic acids within its structure is said to be "self-hybridized."

As used herein, the term " T_m" is used in reference to the "melting temperature." The melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands. A "calculated T_m" refers to a melting temperature determined by calculation from the physical sequence of complementary nucleic acids, along with factors of reaction conditions (e.g., salt concentration, concentrations of the complementary strands in a mixture). Several equations for calculating the T_m of nucleic acids are well known in the art. As indicated by standard references, a simple estimate of the T_m value may be calculated by the equation: T_m = 81.5 + 0.41(% G + C), when a nucleic acid is in aqueous solution at 1 M NaCl (See, e.g., Young and Anderson, (1985) in Nucleic Acid Hybridisation: A Practical Approach (Hames & Higgins, Eds.) pp 47-71, IRL Press, Oxford). Other computations for calculating T_m are known in the art and take structural and environmental, as well as sequence characteristics into account (See, e.g., Allawi, H.T. and SantaLucia, J., Jr.

Biochemistry 36, 10581-94 (1997)) and SantaLucia, Proc Natl Acad Sci U S A.,

95(4): 1460 (1998)).

As used herein, the term "breathing" as used in reference to nucleic acid duplexes, e.g., duplex DNA, refers to spontaneous and reversible denaturation of a portion of a double helix without complete disassociation of the strands. DNA breathing may produce, e.g., a bifurcated end on a duplex, a flap, or a bubble structure.

As used herein, the term "INVADER assay reagents" refers to one or more reagents for detecting target sequences, said reagents comprising nucleic acid molecules capable of participating in formation of an invasive cleavage structure in the presence of the target sequence. In some embodiments, INVADER assay reagents comprise all of the nucleic acid molecules needed to form an invasive cleavage structure in preformed configuration, while in some embodiments, INVADER assay reagents provide or, are used in conjunction with one or more additional reagents {e.g. , primers, polymerizing enzymes, ligases, nucleases) that allow the formation of nucleic acid molecules used in forming an invasive cleavage structure.

In some embodiments, the INVADER assay reagents further comprise an agent for detecting the presence of an invasive cleavage structure {e.g., a cleavage agent). In some embodiments, the nucleic acid molecules comprise first and second

oligonucleotides, said first oligonucleotide comprising a 5' portion complementary to a first region of the target nucleic acid and said second oligonucleotide comprising a 3' portion and a 5' portion, said 5' portion complementary to a second region of the target nucleic acid downstream of and contiguous to the first portion. In some embodiments, the 3' portion of the second oligonucleotide comprises a 3' terminal nucleotide not complementary to the target nucleic acid. In preferred embodiments, the 3' portion of the second oligonucleotide consists of a single nucleotide not complementary to the target nucleic acid. INVADER assay reagents may be found, for example, in U.S. Patent Nos. 5,846,717; 5,985,557; 5,994,069; 6,001 ,567; 6,913,881 ; and 6,090,543, WO 97/27214, WO 98/42873, U.S. Pat. Publ. Nos. 20050014163, 20050074788, 2005016596,

20050186588, 20040203035, 20040018489, 20050164177, and 20090253142; U.S. Pat. Appln. Ser. No. 1 1/266,723; and Lyamichev et al, Nat. Biotech., 17:292 (1999), Hall et al, PNAS, USA, 97:8272 (2000), each of which is herein incorporated by reference in its entirety for all purposes.

As used herein, a "solid support" is any material that maintains its shape under assay conditions, and that can be separated from a liquid phase. Supports that maintain their shape need not be rigid. Indeed, it is contemplated that flexible polymers such as carbohydrate chains, may be used as solid supports, so long as they can be separated from a liquid phase. The present invention is not limited by the type of solid support utilized. Indeed, a variety of solid supports are contemplated to be useful in the present invention including, but not limited to, a bead, planar surface, controlled pore glass (CPG), a wafer, glass, silicon, diamond, graphite, plastic, paramagnetic bead, magnetic bead, latex bead, superparamagnetic bead, plurality of beads, micro fluidic chip, a silicon chip, a microscope slide, a microplate well, a silica gel, a polymeric membrane, a particle, a derivatized plastic film, a glass bead, cotton, a plastic bead, an alumina gel, a

polysaccharide, polyvinylchloride, polypropylene, polyethylene, nylon, Sepharose, poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose, diazocellulose or starch, polymeric microparticle, polymeric membrane, polymeric gel, glass slide, styrene, multi-well plate, column, microarray, latex, hydrogel, porous 3D hydrophilic polymer matrix (e.g., HYDROGEL, Packard Instrument Company, Meriden, Conn.), fiber optic bundles and beads (e.g., BEAD ARRAY (Illumina, San Diego, CA.), described in U.S. Pat. App. 20050164177), small particles, membranes, frits, slides, micromachined chips, alkanethiol-gold layers, non-porous surfaces, addressable arrays, and polynucleotide-immobilizing media (e.g., described in U.S. Pat. App. 20050191660). In some embodiments, the solid support is coated with a binding layer or material (e.g. , gold, diamond, or streptavidin).

In some embodiments one or more of the assay reagents or assay reagent components may be provided in a predispensed format (e.g. , premeasured for use in a step of the procedure without re-measurement or re-dispensing). In some embodiments, selected assay reagent components are mixed and predispensed together. In preferred embodiments, predispensed assay reagent components are predispensed and are provided in a reaction vessel (e.g., including, but not limited to, a reaction tube or a well (e.g., a microtiter plate)). In certain preferred embodiments, the assay reagents are provided in microfluidic devices such as those described in U.S. Pats., 6,627,159; 6,720,187;

6,734,401; and 6,814,935, as well as U.S. Pat. Pub. 2002/0064885, each of which is herein incorporated by reference in its entirety. In particularly preferred embodiments, predispensed INVADER assay reagent components are dried down (e.g. , desiccated or lyophilized) in a reaction vessel.

In some embodiments, the assay reagents or assay reagent components are provided as a kit. As used herein, the term "kit" refers to any delivery system for delivering materials. In the context of reaction assays, such delivery systems include systems that allow for the storage, transport, or delivery of reaction reagents (e.g., oligonucleotides, enzymes, etc. in the appropriate containers) and/or supporting materials (e.g., buffers, written instructions for performing the assay etc.) from one location to another. For example, kits include one or more enclosures (e.g. , boxes) containing the relevant reaction reagents and/or supporting materials. As used herein, the term

"fragmented kit" refers to delivery systems comprising two or more separate containers that each contains a subportion of the total kit components. The containers may be delivered to the intended recipient together or separately. For example, a first container may contain an enzyme for use in an assay, while a second container contains

oligonucleotides. The term "fragmented kit" is intended to encompass kits containing Analyte-specific reagents ' regulated under section 520(e) of the Federal Food, Drug, and Cosmetic Act, but are not limited thereto. Indeed, any delivery system comprising two or more separate containers that each contains a subportion of the total kit components are included in the term "fragmented kit." In contrast, a "combined kit" refers to a delivery system containing all of the components of a reaction assay in a single container (e.g., in a single box housing each of the desired components). The term "kit" includes both fragmented and combined kits.

In some embodiments, the present invention provides assay reagent kits comprising one or more of the components necessary for practicing the present invention. For example, the present invention provides kits for storing or delivering the enzymes and/or the reaction components necessary to practice an assay. The kit may include any and all components necessary or desired for assays including, but not limited to, the reagents themselves, buffers, control reagents (e.g., tissue samples, positive and negative control target oligonucleotides, etc.), solid supports, labels, written and/or pictorial instructions and product information, inhibitors, labeling and/or detection reagents, package environmental controls (e.g., ice, desiccants, etc.), and the like. In some embodiments, the kits provide a sub-set of the required components, wherein it is expected that the user will supply the remaining components. In some embodiments, the kits comprise two or more separate containers wherein each container houses a subset of the components to be delivered. For example, a first container (e.g. , box) may contain an enzyme (e.g. , structure-specific cleavage enzyme in a suitable storage buffer and container), while a second box may contain oligonucleotides (e.g. , oligonucleotides, probe oligonucleotides, control target oligonucleotides, etc.).

In some preferred embodiments, the assay reagents further comprise reagents for detecting a nucleic acid cleavage product. In some embodiments, one or more oligonucleotides in the assay reagents comprise a label. In some preferred embodiments, said first oligonucleotide comprises a label. In other preferred embodiments, said third oligonucleotide comprises a label. In particularly preferred embodiments, the reagents comprise a first and/or a third oligonucleotide labeled with moieties that produce a fluorescence resonance energy transfer (FRET) effect.

As used herein, the term "label" refers to any moiety (e.g. , chemical species) that can be detected or can lead to a detectable response. In some preferred embodiments, detection of a label provides quantifiable information. Labels can be any known detectable moiety, such as, for example, a radioactive label (e.g. , radionuclides), a ligand (e.g. , biotin or avidin), a chromophore (e.g. , a dye or particle that imparts a detectable color), a hapten (e.g., digoxgenin), a mass label, latex beads, metal particles, a

paramagnetic label, a luminescent compound (e.g., bio luminescent, phosphorescent or chemiluminescent labels) or a fluorescent compound.

A label may be joined, directly or indirectly, to an oligonucleotide or other biological molecule. Direct labeling can occur through bonds or interactions that link the label to the oligonucleotide, including covalent bonds or non-covalent interactions such as hydrogen bonding, hydrophobic and ionic interactions, or through formation of chelates or coordination complexes. Indirect labeling can occur through use of a bridging moiety or "linker", such as an antibody or additional oligonucleotide(s), which is/are either directly or indirectly labeled.

Labels can be used alone or in combination with moieties that can suppress (e.g., quench), excite, or transfer (e.g., shift) emission spectra (e.g., fluorescence resonance energy transfer (FRET)) of a label (e.g., a luminescent label).

As used herein, the term "FRET" refers to fluorescence resonance energy transfer, a process in which moieties (e.g., fluorophores) transfer energy e.g., among themselves, or, from a fluorophore to a non-fluorophore (e.g., a quencher molecule). In some circumstances, FRET involves an excited donor fluorophore transferring energy to a lower-energy acceptor fluorophore via a short-range (e.g., about 10 nm or less) dipole- dipole interaction. In other circumstances, FRET involves a loss of fluorescence energy from a donor and an increase in fluorescence in an acceptor fluorophore. In still other forms of FRET, energy can be exchanged from an excited donor flurophore to a non- fluorescing molecule (e.g., a quenching molecule). FRET is known to those of skill in the art and has been described (See, e.g., Stryer et al, 1978, Ann. Rev. Biochem., 47:819; Selvin, 1995, Methods EnzymoL, 246:300; Orpana, 2004 Biomol Eng 21 , 45-50; Olivier, 2005 Mutant Res 573, 103-1 10, each of which is incorporated herein by reference in its entirety).

As used herein, the term "unlabeled" as used in reference to a probe

oligonucleotide refers to a probe oligonucleotide that does not comprise any non-nucleic acid moiety, e.g., a chromorphore or fluorophore, to facilitate detection. An unlabeled probe may comprise modifications, such as 3' blocking groups to prevent extension by a polymerase. As used herein, the term "donor" refers to a moiety (e.g., a fluorophore) that absorbs at a first wavelength and emits at a second, longer wavelength. The term

"acceptor" refers to a moiety such as a fluorophore, chromophore, or quencher and that is able to absorb some or most of the emitted energy from the donor when it is near the donor group (typically between 1-100 nm). An acceptor may have an absorption spectrum that overlaps the donor's emission spectrum. Generally, if the acceptor is a fluorophore, it then re-emits at a third, still longer wavelength; if it is a chromophore or quencher, it releases the energy absorbed from the donor without emitting a photon. In some preferred embodiments, alteration in energy levels of donor and/or acceptor moieties are detected (e.g., via measuring energy transfer (e.g., by detecting light emission) between or from donors and/or acceptor moieties). In some preferred embodiments, the emission spectrum of an acceptor moiety is distinct from the emission spectrum of a donor moiety such that emissions (e.g., of light and/or energy) from the moieties can be distinguished (e.g. , spectrally resolved) from each other.

In some embodiments, a donor moiety is used in combination with multiple acceptor moieties. In a preferred embodiment, a donor moiety is used in combination with a non- fluorescing quencher moiety and with an acceptor moiety, such that when the donor moiety is close (e.g., between 1-100 nm, or more preferably, between 1-25 nm, or even more preferably around 10 nm or less) to the quencher, its excitation is transferred to the quencher moiety rather than the acceptor moiety, and when the quencher moiety is removed (e.g., by cleavage of a probe), donor moiety excitation is transferred to an acceptor moiety. In some preferred embodiments, emission from the acceptor moiety is detected (e.g., using wavelength shifting molecular beacons) (See, e.g., Tyagi, et al, Nature Biotechnology 18: 1 191 (2000); Mhlanga and Malmberg, 2001 Methods 25, 463- 471 ; Olivier, 2005 Mutant Res 573, 103-1 10, and U.S. Pat. App. 20030228703, each of which is incorporated herein by reference in its entirety).

Suitable fluorophores include but are not limited to fluorescein, rhodamine, REDMOND RED dye, YAKIMA YELLOW dye, hexachloro-fluorescein, TAMRA dye, ROX dye, Cy3, Cy3.5, Cy5, Cy5.5, and Cy7, 4,4-difiuoro-5,7-diphenyl-4-bora-3a,4a- diaza- -s-indacene-3 -propionic acid, 4,4-difluoro-5,p-methoxyphenyl-4-bora-3a,4a~ diaza-s-indacene-3 -propionic acid, 4,4-difluoro-5-styryl-4-bora-3a,4-adiaz- a-S-indacene- propionic acid, 6-carboxy-X-rhodamine, N,N,N',N'-tetramethyl-6-carboxyrhodamine, Texas Red, eosin, fluorescein, 4,4-difiuoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene- 3-propionic acid, 4,4-difiuoro-5,p-ethoxyphenyl-4-bora-3a,4a-diaza-s-indacene 3- propionic acid and 4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-S-indacene -propionic acid, 6- carboxyfiuorescein (6-FAM), 2',4', 1 ,4,- tetrachlorofiuorescein (TET), 21,4',51,7',1,4- hexachlorofiuorescein (HEX), 2',7'-dimethoxy-4',5'-dichloro-6-carboxyrhodamine (JOE), 2'-chloro-5'-fiuoro-7',8'-fused phenyl- l,4-dichloro-6-carboxyfiuorescein (NED), 2'- chloro-7'-phenyl-l,4-dichloro-6-carboxyfluorescein (VIC), fluorescein isothiocyanate (FITC), 5,6-carboxymethyl fluorescein, Texas red, nitrobenz-2- oxa-l,3-diazol-4-yl (NBD), coumarin, dansyl chloride, amino-methyl coumarin (AMCA), Erythrosin, BODIPY dye, CASCADE BLUE dye, OREGON GREEN dye, pyrene, lissamine, xanthenes, acridines, oxazines, phycoerythrin, QUANTUM DYE, thiazole orange- ethidium heterodimer, and the like. Suitable quenchers include, but are not limited to, cyanine dyes, e.g., Cy3, Cy3.5, Cy5, Cy5.5, and Cy7, rhodamine dyes, e.g., tetramethyl- 6-carboxyrhodamine (TAMRA) and tetrapropano-6- carboxyrhodamine (ROX),

DABSYL dye, DABCYL dye, cyanine dyes, nitrothiazole blue (NTB), anthraquinone, malachite green, nitrothiazole, or nitroimidazole compounds, QSY7 (Molecular Probes, Eugene, OR), ECLIPSE quencher (Nanogen, San Diego, CA), and the like. Analysis of factors such as absorbance and emission spectra of various molecules in selection of pairs or groups of moieties for use in FRET configurations is well known to those of skill in the art.

Detection of labels or a detectable response (e.g. , provided by the labels) can be measured using a multitude of techniques, systems and methods known in the art. For example, a label may be detected because the label provides detectable fluorescence (e.g. , simple fluorescence, FRET, time-resolved fluorescence, fluorescence quenching, fluorescence polarization, etc.), radioactivity, chemiluminescence,

electrochemiluminescence, RAMAN, colorimetry, gravimetry, hyrbridization (e.g., to a sequence in a hybridization protection assay), X-ray diffraction or absorption,

magnetism, enzymatic activity, characteristics of mass or behavior affected by mass (e.g., MALDI time-of-fiight mass spectrometry), and the like. As used herein the term "interactive label" refers to a label having two or more components that interact so as to produce a detectable effect. The interaction is not limited to any particular nature of interaction. The interaction of the label components may be via direct contact, e.g. , a covalent or non-covalent contact between two moieties (e.g., a protein-protein contact, or collisional energy transfer between proximal moieties); it may comprise resonance energy transfer (e.g., between one or more dyes, or between a dye and a quencher moieties); it may comprise a diffusion effect, e.g., wherein the product from a reaction occurring at the site of one label diffuses to the site of another label to create a detectable effect. The components of an interactive label may be the same (e.g., two or more of the same molecule or atom) or they may be different.

A label may be a charged moiety (positive or negative charge) or alternatively, may be charge neutral. Labels can include or consist of nucleic acid or protein sequence, so long as the sequence comprising the label is detectable. In some embodiments, the label is not nucleic acid or protein.

In some embodiments, a label comprises a particle for detection. For example, in some embodiments, the particle is a phosphor particle. An example of a phosphor particle includes, but is not limited to, an up-converting phosphor particle (See, e.g., Ostermayer, Preparation and properties of infrared-to-visible conversion phosphors. Metall. Trans. 752, 747-755 (1971)). In some embodiments, rare earth-doped ceramic particles are used as phosphor particles. Phosphor particles may be detected by any suitable method, including but not limited to up-converting phosphor technology (UPT), in which up-converting phosphors transfer low energy infrared (IR) radiation to high- energy visible light. Although an understanding of the mechanism is not necessary to practice the present invention and the present invention is not limited to any particular mechanism of action, in some embodiments the UPT up-converts infrared light to visible light by multi-photon absorption and subsequent emission of dopant-dependent phosphorescence (See, e.g., U.S. Patent No. 6,399,397; van De Rijke, et al, Nature Biotechnol. 19(3):273-6 (2001); Corstjens, et al, IEE Proc. Nanobiotechnol. 152(2):64 (2005), each incorporated by reference herein in its entirety.

As used herein, the term "distinct" in reference to signals (e.g., of one or more labels) refers to signals that can be differentiated one from another, e.g., by spectral properties such as fluorescence emission wavelength, color, absorbance, mass, size, fluorescence polarization properties, charge, etc., or by capability of interaction with another moiety, such as with a chemical reagent, an enzyme, an antibody, etc.

As used herein, the term "synthetic" as used in reference to a polynucleotide or oligonucleotide {e.g., a probe) refers to a nucleic acid created in a cell-free in vitro reaction, e.g., an enzymatic or chemical synthesis reaction. Examples of enzymatic formation of a synthetic nucleic acid include formation by restriction digestion, polymerization (templated or non-templated), ligation, etc. Examples of chemical synthesis of nucleic acid include but are not limited to, e.g., phosphodiester and phosphotriester chemistries, phosphoramidite and H-phosphonate, chemistries, etc. See e.g., Methods in Molecular Biology, Vol 20 : Protocols for Oligonucleotides and Analogs pp. 165-189 (S. Agrawal, Ed., Humana Press, 1993).; Oligonucleotides and Analogues: A Practical Approach , pp. 87-108 (F. Eckstein, Ed., 1991); and Uhlmann and Peyman, supra. Agrawal and Iyer, Curr. Op. in Biotech. 6: 12 (1995); and Anti-sense Research and Applications (Crooke and Lebleu; Eds., CRC Press, Boca Raton, 1993), Beaucage and Caruthers, Tetrahedron Lett. 22: 1859-1862 (1981), and Agrawal and Zamecnik, U.S. Pat. No. 5,149,798 (1992). In some embodiments, synthetic oligonucleotides are introduced into a reaction pre-formed, while in some embodiments, synthetic

oligonucleotides are formed or modified within the reaction, e.g. , by action of a polymerase, ligase, cleavage enzyme, or the like.

As used herein, the term "FEN-1" in reference to an enzyme refers to a non- polymerase flap endonuclease from a eukaryote or archaeal organism.

As used herein, the term FEN-1 activity refers to any enzymatic activity of a FEN-1 enzyme, including but not limited to flap endonuclease (FEN), nick exonuclease (EXO), and gap endonuclease (GEN) activities (see, e.g., Shen, et al., BioEssays Volume 27, Issue 7, Pages 717 - 729, incorporated herein by reference).

As used herein, the term "identifying the presence of a polymorphism" refers to any method of inferring the identity of a nucleotide at a position of a suspected point of genetic variation. In some embodiments, the presence of a particular polymorphism or mutation is directly detected, e.g., the presence of the polymorphism causes a detectable event to occur {e.g. , probe hybridization, probe cleavage, nucleic acid target or signal amplification, etc. ), while in other embodiments, the presence of a polymorphism or mutation may be inferred from the absence of a particular nucleotide or sequence of nucleotides {e.g., the absence of a wild-type nucleotide in a position in a nucleic acid sequence as an indicator of the presence of a mutant or polymorphic nucleotide at that position.)

As used herein, the term "determining an identity of an organism" encompasses any manner of assigning identification to an organism of interest, including but not limited to identification of a unique, individual organism, e.g., as a variant among a population of related organisms, and/or classification of an organism, e.g., by species, genus, family order, etc. Identity of an organism may be by phenotype or genotype.

DESCRIPTION OF THE INVENTION

The present invention relates to assays for the detection of nucleic acid. In accordance with the present invention, novel methods, reaction mixtures, compositions and kits are provided for the production of specific nucleic acid molecules called priming oligonucleotides, for use in assays for the detection and/or quantitation of nucleic acid target sequences.

Embodiments of the present invention provide assays in which key assay reaction oligonucleotides are created enzymatically during the assay reaction.

In some embodiments the detection assay provides detection of nucleic acids in which a target-dependent "priming" oligonucleotide is generated by the action(s) of a polymerase enzyme and/or a cleavage enzyme. Once generated, the priming

oligonucleotide can be detected by various methods. In one embodiment, for example, a method uses the priming oligonucleotide to generate a unique flap or arm molecule using the highly-specific structure recognition and cleavage by a FEN-1 endonuclease, e.g., an archaeal FEN-1.

In some embodiments, many copies of the released arm are produced in a reaction comprising annealing the priming oligonucleotide to an extension/cleavage template oligonucleotide ("ECT" oligonucleotide), followed by a cycles of extension, cleavage and dissociation, e.g., as shown schematically in the embodiment diagrammed in Figure 6. This results in the generation of many copies of a detectable flap or arm sequence per priming oligonucleotide. The arm sequence can be universal with respect to target (i.e., the sequence is not related to the target and can be re -used in many different target detection assay designs) and can be detected in multiple ways, e.g., by the cleavage of a FRET cassette such as is shown in Figure 1 A. In another embodiment, the priming oligonucleotide may anneal to a secondary reaction target to direct cleavage of a FRET probe, e.g., using a structure similar to the Primary Reaction structure shown in Panel a of Figure 1A.

The embodiments disclosed herein for Enzyme Mediated Arm Generation INvader assays are referred to collectively as "EMAGIN" assays. The EMAGIN assays as described herein address the background signal generation in cleavage assays such as the INVADER assay. As discussed above, a significant source of background signal is fragmented oligonucleotides present as part of the synthetically-made probe

oligonucleotide preparation. It is readily appreciated that broken probe fragments (e.g., as shown in Primary Reaction structure shown in Panel a of Figure 1A) may include molecules that are the same as the flap (aka "arm") sequence produced during the detection assay. This arm sequence is a design component required for signal generation, e.g., with a FRET cassette. As shown in Figure 1 A, the arm is cleaved from the probe oligonucleotide by the 5' nuclease or flap endonuclease enzyme when the target sequence of interest is present in the reaction. The released arm forms a preferred substrate for the 5' nuclease enzyme with a FRET cassette (or other secondary reaction components) provided for detection. Cleavage of the arm/FRET cassette structure results in the generation of a detectable signal that is indicative of the presence of the target nucleic acid.

Background signal generated in the reaction is caused by cleavage of the FRET cassette oligonucleotide due to structures formed with arm sequences present in the reaction due to synthesis by-products, impurities from the probe oligonucleotide and cleavage of a non-preferred substrate referred to as the X-structure, which forms between the arm sequence present on a non-cleaved probe oligonucleotide and a FRET cassette.

To avoid reliance on a species of reaction product that is also found in the oligonucleotide preparations prior to use, the EMAGIN assay entails two general processes: 1) target-dependent generation of a priming oligonucleotide, and 2) detection of the unique priming oligonucleotide.

In certain embodiments provided herein, a priming oligonucleotide is a nucleic acid sequence with a defined composition region (DCR) and a distinct 3 '-end (D3E) that is generated in the presence of a specific target nucleic acid. In some embodiments, a priming oligonucleotide is generated by extension of a target-specific primer, with displacement and cleavage of the extended primer based on structure recognition to produce the priming oligonucleotide. In some embodiments, the extension is conducted with a reverse transcriptase enzyme.

In some embodiments, the creation of the priming oligonucleotide may comprise recognition (e.g., binding, extension and/or cleavage) of existing short nucleic acid sequences (e.g. microRNAs), or may comprise restriction digestion of DNA and subsequent recognition of a specific fragment or nucleic acid end produced by the restriction enzyme. In yet other embodiments, production of a priming oligonucleotide may comprise PCR and/or cleavage of an amplicon based on structure recognition. In yet other embodiments, the creation of a priming oligonucleotide may comprise controlled extension of a primer on DNA or RNA target template, e.g., by use of a blocking oligonucleotide or a restricted pool of nucleoside triphosphates (e.g., lacking one or more NTP/dNTPs necessary for unrestricted synthesis).

Figure IB diagrams one embodiment comprising use of a 5' nuclease cleavage to form a priming oligonucleotide having distinct 3 'end. As diagrammed, a primer is extended on a target nucleic acid to produce an extension product having both primer- originated sequence and target-specific sequence. The extension product is used to form a cleavage structure with an oligonucleotide, e.g., an "IT" oligonucleotide that serves as both an "INVADER" oligonucleotide and a pseudo-target strand in forming an invasive cleavage structure. Cleavage of the structure with a 5' nuclease, e.g., a FEN-1

endonuclease within the target-specific portion of the extension product produces a new oligonucleotide species, termed a "priming oligonucleotide" that has a distinct 3' end that does not exist on any of the chemically synthesized molecules provided in the assay set- up. This unique new molecule can be used or detected by any method, including the methods described herein below. The extended primer product used to produce the priming oligonucleotide may be separated from its target-derived template strand by a number of different methods. In some embodiments, an increase in temperature is used to disassociate the extended primer from the template strand, as diagrammed in Figure 2. In some embodiments, the temperature is cycled, e.g., to allow additional unextended primers to hybridize to the template and be extended, such that multiple copies of the extended primer are produced from each template or target molecule.

In some embodiments, the extended primer is removed from the template by strand displacement. For example, in some embodiments, two primers may be configured to anneal to the target strand, such that a downstream primer forms the extended primer product used to produce the priming oligonucleotide and the upstream primer is extended to use the action of a displacing polymerase to displace the downstream extended product strand from the target, as shown in the embodiment diagrammed in Figure 3. In certain preferred embodiments, displacement of the extended primer from the template strand is done isothermally.

In some embodiments, reactions are configured to permit invasion of the extended primer/template duplex by an oligonucleotide, e.g., an "IT" oligonucleotide as diagrammed in Figure 4. Conditions favorable for breathing of nucleic acid duplexes include e.g., elevated temperature and/or the presence of osmolytes such as betaine and/or trimethylamine oxide. DNA breathing may also be enhanced at particular regions of sequence, e.g., A/T rich regions, or in duplexes comprising mismatched bases. See, e.g., US Pat. Pub. 2007/0054301, which is incorporated herein by reference.

The extended primer, once at least partially displaced from the target strand, can be cleaved, resulting in a priming oligonucleotide having a distinctive 3' end. One created, the existence of the new species of oligonucleotide can be detected by any method for detecting a specific oligonucleotide sequence. US Patent Nos. 5,994,069 and 8,206,904, both incorporated herein by reference in their entireties, for example, provide a number of diverse methods for specific detection of small nucleic acid molecules, such as released flap sequences. It is contemplated that any one of the methods may find application in embodiments of the present invention. Use of the priming oligonucleotide approach offers particular advantages. For example, in assay that depend on having highly pure probe oligonucleotides to minimize background from damaged or broken probes, purification of the oligonucleotides involved in a signal amplification are typically purified to a very high degree, e.g., though electrophoresis or HPLC. This contrasts with general methods of treating primer oligonucleotides for use, e.g., in PCR. Amplification primers typically need not be as pure as the signal-generating oligonucleotides, and often are minimally processed prior to use. The priming oligonucleotide approach disclosed herein uses the discriminatory power of the polymerase and nuclease enzymes to selectively modify primers having the correct sequence and structure. Because the enzymes can distinguish between correct oligonucleotides and reaction by-products, the primers of the method may be used with minimal processing.

EXPERIMENTAL

The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.

EXAMPLE 1

Strand Invasion by Snaking Oligonucleotides

Tests were conducted to evaluate the ability of different snake test

oligonucleotides (3369-05 oligonucleotides) to direct cleavage of a probe oligonucleotide with Afu FEN- 1.

Mixtures of probe, FRET cassette, Afu FEN-1, and different 3369-05

oligonucleotides shown in Figures 7 and 8 were tested. Each 20 μΐ reaction included 200 ng of Afu FEN-1 endonuclease. 0.5 μΜ probe, 0.25μΜ FRET cassette and 0.05 or 0.005 μΜ 3369-05 oligonucleotide in a buffer of 10 mM MOPS with either 7.5 mM MgCl₂ or a combination of 25 mM KC1 with 2 mM MgCl₂. The reactions are assembled in a reaction plate as follows: Reaction lOmM MOPS, 25mM KC1,

lOmM MOPS, 7.5mM MgC12

Buffer 2mM MgC12

3369-05

0.00 5μΜ olig3 [ ] 0.05 μΜ 0.00 5μΜ 0.05 μΜ

1 1 z j o 7 o o a none no ne no ne no ne none no ne no ne none b 3369-05-01 3369-05-07 3369-05-01 3369-05-07 3369-05-01 3369-05-07 3369-05-01 3369-05-07 c 3369-05-02 3369-05-08 3369-05-02 3369-05-08 3369-05-02 3369-05-08 3369-05-02 3369-05-08 d 3369-05-03 3369-05-09 3369-05-03 3369-05-09 3369-05-03 3369-05-09 3369-05-03 3369-05-09 e 3369-05-04 3369-05-10 3369-05-04 3369-05-10 3369-05-04 3369-05-10 3369-05-04 3369-05-10 f 3369-05-05 3369-05-11 3369-05-05 3369-05-11 3369-05-05 3369-05-11 3369-05-05 3369-05-11 g 3369-05-06 3369-05-12 3369-05-06 3369-05-12 3369-05-06 3369-05-12 3369-05-06 3369-05-12

The reactions were incubated at 55°C, 59°C, and 63°C for 30 minutes.

Fluorescence was read and results were analyzed to assess cleavage of the FRET cassette. The results are shown in Figure 9. The results demonstrate that a snake structure will form as defined by the 3 '-end snaking sequences of the ECT and generate cleavage at a defined location. The snake structure is able to form by displacing partial to full complementary sequences. The presence, number and location of mismatches have an effect on the efficiency of the structure formation and/or subsequent cleavage.

EXAMPLE 2

Strand Invasion by Snaking Oligonucleotides II

Tests were conducted to evaluate the ability of a combination of a DNA polymerase (Vent) and Afu FEN-1 endonuclease to extend a priming oligonucleotide on an ECT oligonucleotide and subsequently cleave the extended priming oligonucleotide. The effect of the length of the priming oligonucleotide and different mismatches with respect to the ECT oligonucleotide, along with the effect of the length of snaking sequences were also evaluated. A mixture of FRET cassette, Afu FEN-1 endonuclease, and DNA polymerase different ECT oligonucleotides as shown in Figure 10 and priming oligonucleotides as shown in Figure 11 were incubated together at different temperatures.

Each 20 μΐ reaction contained 100 ng Afu FEN-1 endonuclease and 0.5 units of Vent Exo- DNA polymerase (New England Biolabs), 0.025mM each dNTP (dATP, dCTP, dGTP and dTTP), 0.01 μΜ of a priming oligonucleotide, 0.25μΜ FRET cassette and 0.25 μΜ ECT oligonucleotide in a buffer of 10 mM MOPS with 7.5 mM MgC12. The reactions are assembled in a reaction plate as follows:

The reactions were incubated at 57°C for 60 minutes then 63°C for 30 minutes or at 61°C for 90 minutes. Fluorescence was read and results were analyzed to determine assess cleavage of the FRET cassette.

The results are shown in Figure 12. These data show that snaking sequences of 3, 4, 5, or 6 nucleotides give very similar results with different priming oligonucleotide sequences. These data show that the length of the priming oligonucleotide and the number and positions of mismatches have an effect on the amount of signal produced in the reactions.

EXAMPLE 3

Strand Invasion by Snaking Oligonucleotides III

Tests were conducted to evaluate the effects of using different DNA polymerases. Mixtures of FRET cassette, Afu FEN-1 endonuclease, a DNA polymerase, and different ECT oligonucleotides as shown in Figure 10 and priming oligonucleotides as shown in Figure 11 were incubated together at different temperatures.

Each 20 μΐ reaction contained 100 ng Afu FEN-1 endonuclease and 0.5 units of one of Vent Exo- DNA polymerase (New England Bio labs), cloned Pfu DNA

Polymerase, Platinum Tfi DNA polymerase, Platinum Tfi Exo- DNA polymerase, GoTaq DNA polymerase or Tfi DNA polymerase; 0.025mM each dNTP (dATP, dCTP, dGTP and dTTP), 0.01 μΜ of a priming oligonucleotide, 0.25μΜ FRET cassette and 0.25 μΜ ECT oligonucleotide in a buffer of 10 mM MOPS with 7.5 mM MgCl₂. GoTaq and Tfi DNA polymerases were also tested in the absence of FEN-1 endonuclease. The reactions are assembled in a reaction plate as follows:

The reactions were incubated at either 57°C or 61°C for 180 minutes.

Fluorescence was read and results were analyzed to determine assess cleavage of the FRET cassette. The results are shown in Figures 13-16. The results demonstrate that different polymerases can be used for the ECT cascade reaction. The results indicate that priming oligonucleotide length and position of mismatches have an effect on the signal accumulation that is dependent on the DNA polymerase. All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described compositions and methods of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the relevant arts are intended to be within the scope of the following claims.

Claims

WE CLAIM:

1. A method of detecting a target nucleic acid, comprising:

c) detecting said detectable structure.

2. The method of claim 1, wherein forming a priming oligonucleotide comprises extending a primer on said target nucleic acid with a polymerase to form an extended primer having an extension region.

3. The method of claim 2, further comprising cleaving said extended primer in said extension region to form said priming oligonucleotide.

4. The method of claim 1, wherein forming said detectable structure comprises extending said priming oligonucleotide with a template-dependent polymerase to form an extended priming oligonucleotide having an extension region.

5. The method of claim 1 or claim 4, wherein said detectable structure comprises a cleavage structure for a structure-specific nuclease.

6. The method of claim 5, wherein said cleavage structure is an invasive cleavage structure.

7. The method of claim 6, wherein said oligonucleotide template is an

extension/cleavage template having a 3' portion, and wherein said invasive cleavage structure forms when said 3' portion of said extension/cleavage template displaces one or more nucleotides of the extended priming oligonucleotide duplexed to said

extension/cleavage template.

8. The method of claim 5, wherein said detecting comprises cleaving said cleavage structure.

9. The method of claim 8, wherein said cleaving said cleavage structure comprises cleaving said extended priming oligonucleotide.

10. The method of claim 9, wherein cleaving said extended priming oligonucleotide comprising cleaving the extension region off said extended priming oligonucleotide to release a priming oligonucleotide.

11. The method of claim 9, wherein cleaving said extended priming oligonucleotide produces a released arm oligonucleotide.

12. The method of claim 11, wherein said detecting comprises hybridizing said released arm oligonucleotide to a FRET cassette to form a detectable structure.

13. The method of claim 11, wherein said detectable structure is an invasive cleavage structure and wherein said detecting comprises cleaving said FRET cassette.

14. The method of Claim 1, wherein said detecting comprises detection selected from the group consisting of detection of fluorescence, mass, fluorescence energy transfer, radioactivity, luminescence, phosphorescence, fluorescence polarization, and charge.

15. The method of Claim 1, wherein said reaction comprises a 5' nuclease.

16. The method of Claim 15, wherein said 5' nuclease comprises a FEN-1 endonuclease from an archaeal species.

17. The method of Claim 2, wherein said polymerase comprises a DNA polymerase from Bacillus stearothermophilus.

18. The method of Claim 1 , wherein said target nucleic acid is selected from the group consisting of DNA and RNA.

19. The method of Claiml, wherein said reaction comprises an osmolyte.

20. The method of claim 19, wherein said osmolyte is selected from betaine and trimethylamine N-oxide.

21. A method of detecting a target nucleic acid, comprising:

a) incubating said target nucleic acid under conditions wherein: i) a primer is extended on said target nucleic acid to form an extended primer;

ii) said extended primer is modified to form a priming oligonucleotide comprising a distinctive 3' end;

iii) said priming oligonucleotide hybridizes to an

extension/cleavage template oligonucleotide and is extended to form an extended priming oligonucleotide;

iv) a snaking region of said extension/cleavage template oligonucleotide forms a cleavage structure comprising said extended priming oligonucleotide;

v) said extended priming oligonucleotide is cleaved to form a cleavage product; b) detecting cleavage of said extended priming oligonucleotide.

22. The method of claim 21, wherein said extended primer is modified by cleaving.

23. The method of claim 22, wherein said cleaving is cleaving by a structure specific nuclease.

24. The method of claim 23, wherein said structure specific nuclease is a FEN-1 endonuclease.

25. The method of claim 21 , wherein said primer is extended by a DNA polymerase.

26. The method of claim 25, wherein said DNA polymerase has a strand- displacement activity.

27. A composition for detecting a target nucleic acid comprising:

i) a priming oligonucleotide comprising a distinctive 3' end;

iv) a structure-specific nuclease activity.

28. The composition of Claim 27, wherein said polymerase activity is provided as a DNA or RNA polymerase.

29. The composition of Claim 27, wherein said structure specific nuclease activity is a 5' nuclease activity.

30. The composition of claim 29, wherein said 5' nuclease is activity is provided as FEN-1 endonuclease and/or a DNA polymerase.

31. The composition of claim 27, wherein said polymerase activity and said structure specific nuclease activity are provided by a single enzyme.

32. A kit for forming a priming oligonucleotide, comprising:

i) a primer;

ii) an extension/cleavage template oligonucleotide comprising a region complementary to at least a portion of said priming oligonucleotide and a snaking region configured to form a cleavage structure when a priming oligonucleotide is extended by a template-dependent polymerase;

iii) a polymerase activity; and

iv) a structure-specific nuclease activity.

33. The kit of claim 32, further comprising a FRET cassette.

34. The kit of Claim 32, wherein said structure specific nuclease activity is provided as a FEN-1 endonuclease.