WO2005068660A1 - Solid-phase multiplexed invader assay - Google Patents
Solid-phase multiplexed invader assay Download PDFInfo
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- WO2005068660A1 WO2005068660A1 PCT/US2003/040973 US0340973W WO2005068660A1 WO 2005068660 A1 WO2005068660 A1 WO 2005068660A1 US 0340973 W US0340973 W US 0340973W WO 2005068660 A1 WO2005068660 A1 WO 2005068660A1
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- nucleic acid
- target nucleic
- cleavage
- oligonucleotide
- oligonucleotides
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- 238000010839 reverse transcription Methods 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 239000002336 ribonucleotide Substances 0.000 description 1
- 125000002652 ribonucleotide group Chemical group 0.000 description 1
- 229920002477 rna polymer Polymers 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000002000 scavenging effect Effects 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
Definitions
- BACKGROUND Assay methods have been described which are useful for the detection and characterization of specific target nucleic acids and sequence variations within such specific nucleic acids, including single nucleotide polymorphisms or SNPs.
- One such assay has been described, which utilizes nucleic acid cleavage structures that are cleaved in a site-specific manner to indicate the presence of specific nucleic acid sequences. See Brow, et al, U.S. Patent No. 5,846,717 and Hall, et al., U.S. Patent No. 5,994,069.
- Such methods have great utility in the detection and diagnosis of various genetic diseases and disorders, in the identification of gene mutations, and in many other contexts, as will be appreciated by the skilled artisan.
- multiplexed assays have been developed using, for example, multiple fluorescent labels, such methods have a number of limitations, including increased cost and a limit on the number of suitable labels that can be used simultaneously in multiplexed assays. What is needed, therefore, is an assay method that can detect and characterize multiple specific target nucleic acids and/or sequence variations in a single reaction (a "multiplexed" assay) that is efficient, reliable, and cost effective.
- the invention includes a method for detecting the presence of a target nucleic acid comprising: (a) providing: (i) a sample comprising at least one target nucleic acid having a first portion, a second portion, and a third portion, wherein the first portion is downstream of and contiguous to the second portion, the second portion is downstream of and contiguous to the third portion, and wherein each target nucleic acid is associated with a preselected bar code sequence; (ii) an invading oligonucleotide, having a 3' portion and a 5' portion, the 5' portion containing sequence complementary to the first portion of the target nucleic acid and the 3' portion containing sequence complementary to the second portion of the target nucleic acid; (iii) a primary cleavage oligonucleotide, each primary cleavage oligonucleotide having a 3' portion complementary to the third portion of the target nucleic acid, a cle
- the invention includes a method for detecting and identifying a single nucleotide polymorphism (SNP) in a target nucleic acid molecule in an assay, comprising: (a) providing: (i) at least one target nucleic acid molecule having a first portion, a second portion comprising a SNP, and a third portion, wherein the first portion is downstream of and continguous to the second portion, the second portion is downstream of and contiguous to the third portion; (ii) an invading oligonucleotide, having a 3' portion and a 5' portion, the 5' portion of the invading oligonucleotide containing sequence complementary to the first portion of the target nucleic; (iii) a set of primary cleavage oligonucleotides, each primary cleavage oligonucleotide having a 3' portion complementary to the third portion of the target nucleic acid, a middle portion containing a nucleot
- the invention includes a method for detecting and identifying a single nucleotide polymorphism at a specific gene locus in a target nucleic acid molecule in an assay, comprising: (a) providing: (i) at least one target nucleic acid molecule having a first portion, a second portion comprising a SNP, and a third portion, wherein the first portion is downstream of and continguous to the second portion, the second portion is downstream of and contiguous to the third portion; (ii) an invading oligonucleotide, having a 3' portion and a 5' portion, the 5' portion of the invading oligonucleotide containing sequence complementary to the first portion of the target nucleic; (iii) at least one set of primary cleavage oligonucleotides for each gene locus, each primary cleavage oligonucleotide having a 3' portion complementary to the third portion of the target nucleic acid, a middle
- the invention includes a method of detecting and identifying a single nucleotide polymorphism (SNP) in a target nucleic acid molecule in an assay, comprising: providing: (i) a target nucleic acid molecule having a first portion, a second portion comprising a SNP, and a third portion, wherein the first portion is downstream of and contiguous to the second portion, the second portion is downstream of and contiguous to the third portion; (ii) an invading oligonucleotide, having a 3' portion and a 5' portion, the 5' portion of the invading oligonucleotide containing sequence complementary to the first portion of the target nucleic; (iii) a set of primary cleavage oligonucleotides, each primary cleavage oligonucleotide having a 3' portion complementary to the third portion of the target nucleic acid, a middle portion containing a nucleotide containing
- SNP
- the invention includes a method of detecting and identifying a single nucleotide polymorphism (SNP) and a gene locus in a target nucleic acid molecule in an assay, comprising: (a) providing: (i) a target nucleic acid molecule having a first portion, a second portion comprising a SNP, and a third portion, wherein the first portion is downstream of and contiguous to the second portion, the second portion is downstream of and contiguous to the third portion; (ii) an invading oligonucleotide, having a 3' portion and a 5' portion, the 5' portion of the invading oligonucleotide containing sequence complementary to the first portion of the target nucleic; (iii) at least one set of primary cleavage oligonucleotides for each gene locus, each primary cleavage oligonucleotide having a 3' portion complementary to the third portion of the target nucleic acid,
- the invention includes a method of detecting and identifying a single nucleotide polymorphism (SNP) at a specific locus in a target nucleic acid molecule in an assay, comprising: (a) providing: (i) a target nucleic acid molecule having a first portion, a second portion comprising a SNP, and a third portion, wherein the first portion is downstream of and contiguous to the second portion, the second portion is downstream of and contiguous to the third portion; (ii) an invading oligonucleotide having a 3' portion and a 5' portion, the 5' portion of the invading oligonucleotide containing sequence complementary to the first portion of the target nucleic; (iii) at least one set of primary cleavage oligonucleotides, each primary cleavage oligonucleotide having a 3' portion complementary to the third portion of the target nucleic acid, a middle portion containing
- the invention includes a method of detecting and identifying a single nucleotide polymorphism (SNP) at a specific locus in a target nucleic acid molecule in an assay, comprising: (a) providing: (i) a target nucleic acid molecule having a first portion, a second portion comprising a SNP, and a third portion, wherein the first portion is downstream of the second portion, the second portion is downstream of and contiguous to the third portion; (ii) an invading oligonucleotide having a 3' portion and a 5' portion, the 5' portion of the invading oligonucleotide containing sequence complementary to the first portion of the target nucleic; (iii) a set of primary cleavage oligonucleotides comprising four unique oligonucleotides for each locus of interest, such that each primary cleavage oligonucleotide contains a unique sequence ("bar code") denoting
- the invention includes a method of detecting and identifying a single nucleotide polymorphism (SNP) at a specific locus in a target nucleic acid molecule in an assay, comprising: (a) providing: (i) a target nucleic acid molecule having a first portion, a second portion comprising an SNP, and a third portion, wherein the first portion is downstream of the second portion, the second portion is downstream of and contiguous to the third portion; (ii) an invading oligonucleotide having a 3' portion and a 5' portion, the 5' portion of the invading oligonucleotide containing sequence complementary to the first portion of the target nucleic; (iii) a set of primary cleavage oligonucleotides comprising four unique oligonucleotides for each locus of interest, such that each primary cleavage oligonucleotide contains a unique sequence ("bar code") denoting
- SNP
- the invention includes a method of detecting and identifying a single nucleotide polymorphism (SNP) at a specific locus in a target nucleic acid molecule in an assay, comprising: (a) providing: (i) a target nucleic acid molecule having a first portion, a second portion comprising a SNP, and a third portion, wherein the first portion is downstream of the second portion, the second portion is downstream of and contiguous to the third portion; (ii) an invading oligonucleotide having a 3' portion and a 5' portion, the 5' portion of the invading oligonucleotide containing sequence complementary to the first portion of the target nucleic; (iii) a set of primary cleavage oligonucleotides comprising four unique oligonucleotides for each locus of interest, such that each primary cleavage oligonucleotide contains a unique sequence ("bar code") denoting
- the invention includes a method of monitoring the specific activity of a nucleic acid cleavage means, comprising: (a) providing: (i) a target nucleic acid molecule having a first portion, a second portion, and a third portion, wherein the first portion is downstream of the second portion, the second portion is downstream of and contiguous to the third portion; (ii) an invading oligonucleotide having a 3' portion and a 5' portion, the 5' portion of the invading oligonucleotide containing sequence complementary to the first portion of the target nucleic; (iii) a primary cleavage oligonucleotide, each primary cleavage oligonucleotide having a 5' portion containing a first detectable label and a 3' portion complementary to the third portion of the target nucleic acid and containing a second detectable label; (iv) a nucleic acid cleavage means; (b) mixing the target nucleic acid
- the invention includes a kit for detecting and identifying a single nucleotide polymorphism (SNP) at a specific locus in a target nucleic acid molecule in an assay, the kit comprising: a set of primary cleavage oligonucleotides corresponding to the target nucleic acid comprising four unique oligonucleotides for each locus of interest, such that each primary cleavage oligonucleotide contains a unique sequence ("bar code") denoting a specific locus and a specific SNP, each primary cleavage oligonucleotide having a 3' portion complementary to the target nucleic acid, a middle portion comprising a nucleotide selected from the group consisting of A, T, C, and G, and a 5' portion containing the bar code and a detectable label; an invading oligonucleotide corresponding to the target nucleic acid; a nucleic acid cleavage means for a target nu
- FIG. 1 is a schematic diagram of one embodiment of the system and method for detecting a target nucleic acid molecule of the present invention.
- FIG. 2 is an illustration of a 4x4 array of capture oligonucleotides deposited in a well of an activated polypropylene plate.
- FIG. 3 is a flow chart illustrating a method for analyzing fluorescence changes in capture oligonucleotides deposited onto a solid substrate according to an embodiment of the present invention.
- FIG. 1 is a schematic diagram of one embodiment of the system and method for detecting a target nucleic acid molecule of the present invention.
- FIG. 2 is an illustration of a 4x4 array of capture oligonucleotides deposited in a well of an activated polypropylene plate.
- FIG. 3 is a flow chart illustrating a method for analyzing fluorescence changes in capture oligonucleotides deposited onto a solid substrate according to an embodiment of the present invention.
- FIG. 4 is a schematic diagram of an embodiment of the present invention wherein an assay specific probe is cleaved from a primary cleavage structure and hybridized to a capture oligonucleotide coupled to a fluorescent label and deposited onto a solid substrate.
- FIG. 5 is an illustration of two primary cleavage oligonucleotides, each having both a gene loci-specific bar code sequence and a bar code sequence specific for the nucleotide A at the site of an SNP.
- FIG. 6 is an illustration of two primary cleavage oligonucleotides, each having both an SNP-specific bar code sequence and a bar code sequence specific for gene loci 1.
- FIG. 5 is an illustration of two primary cleavage oligonucleotides, each having both a gene loci-specific bar code sequence and a bar code sequence specific for gene loci 1.
- FIG. 7 is a schematic diagram of an embodiment of the present invention wherein a 5'-labeled assay specific probe is cleaved from a primary cleavage structure and hybridized to a capture oligonucleotide deposited onto a solid substrate.
- FIG. 8 is a schematic diagram illustrating Enzyme-Labeled Fluorescence (ELF) signal amplification according to an embodiment of the present invention using labeled primary cleavage oligonucleotides.
- FIG. 9 is a schematic diagram illustrating the use of a dual-labeled primary cleavage oligonucleotide according to the practice of one embodiment of the present invention.
- FIG. EMF Enzyme-Labeled Fluorescence
- FIG. 10 is a schematic diagram illustrating the use of paramagnetic or magnetic beads and a magnet to scavenge uncleaved affinity-tagged primary cleavage oligonucleotides following a cleavage reaction.
- FIG. 11 is a schematic diagram and photograph showing the effect of primary cleavage oligonucleotide concentration on the effectiveness of the removal of uncleaved reactants.
- FIG. 12 is a schematic diagram and table showing the effectiveness of using biotin-tagged dual labeled primary cleavage oligonucleotide with magnetic bead capture to remove uncleaved reaction product.
- polynucleotide or “nucleic acid” and their respective plurals are used essentially interchangeably herein and are intended to include naturally occurring or synthesized double stranded deoxyribonucleic acid (hereinafter “DNA”), single stranded DNA, or ribonucleic acid (hereinafter “RNA”).
- DNA double stranded deoxyribonucleic acid
- RNA ribonucleic acid
- complementarity refer to polynucleotides that undergo Watson-Crick base pairing. This includes the pairing of "nucleoside analogs", such as deoxyinosine, nucleosides with 2-aminopurine bases, and the like, that may be employed.
- nucleoside includes the natural nucleosides, including 2'-deoxy and 2'-hydroxyl forms, e.g. as described in Kornberg and Baker, DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992).
- "Analogs" in reference to nucleosides includes synthetic nucleosides having modified base moieties and/or modified sugar moieties that are capable of specific hybridization, e.g. described by Scheit, Nucleotide Analogs (John Wiley, New York, 1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990), or the like. Such analogs include synthetic nucleosides designed to enhance binding properties, reduce degeneracy, increase specificity, and the like.
- Total or perfect complementarity means that the polynucleotide or oligonucleotide strands making up a duplex form a double stranded structure with one another such that every nucleotide in each strand undergoes Watson-Crick base pairing with a nucleotide in the other strand and that there are no mismatches.
- "Partially complementary” as used herein includes polynucleotide or oligonucleotide strands containing unmatched and non-hybridizing nucleotides, but in which the remaining "matched" nucleotides undergo Watson-Crick base pairing.
- a “self-complementary” nucleic acid is one in which separate regions of the nucleic acid are capable of base-pairing with one another to for ⁇ i, for example, a hairpin structure.
- “Homology” refers to a degree of identity between two or more nucleic acid sequences. There may be partial homology or complete homology. A partially identical sequence is one that is less than 100% identical to another sequence.
- “Hybridization” is used herein in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization is affected by variables such as the degree of complementary between the nucleic acids, stringency of the hybridization conditions involved, the T (melting temperature) of the formed hybrid, and the G:C ratio within the nucleic acids.
- oligonucleotide as used herein is defined as a molecule comprised of two or more 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. Such oligonucleotides may be generated by various methods, including chemical synthesis, DNA replication, reverse transcription, or a combination thereof.
- 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.
- 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.
- 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 "downstream" region when moving along a strand of nucleic acid in a 5' to 3' direction.
- Target nucleic acid as used herein may comprise single or double-stranded DNA or RNA.
- Target nucleic acids contain a sequence that has at least partial complementarity with at least a primary cleavage oligonucleotide, and may also have at least partial complementarity with an invading oligonucleotide.
- Primary cleavage oligonucleotide refers to an oligonucleotide which contains a flap portion and a non-flap portion, the non-flap portion having a least partial complementarity with the target nucleic acid, and the flap portion optionally having one or more bar code sequences.
- Bar code sequence refers to a specific sequence of nucleotides selected so as to identify a specific feature of the target nucleic acid. Bar code sequences typically are nonsense or unrelated sequence to that of the gene of interest in the target nucleic acid, designed for specific (perfect match) hybridization to a region of the solid phase tethered capture oligonucleotide. Examples include a locus bar code sequence, in which different gene loci are assigned different bar code sequences, and a single nucleotide polymorphism (“SNP”) bar code, in which different SNPs are given different bar code sequences. Other examples of useful bar code sequences will be apparent to one of skill in the art.
- “Invading oligonucleotide” refers to an oligonucleotide which contains sequences at its 3' end which are substantially the same as sequences located at the 5' end of the non- flap portion of a primary cleavage oligonucleotide; these regions will compete for hybridization to the same segment along a complementary target nucleic acid.
- a “capture oligonucleotide” as used herein refers to an oligonucleotide that is complementary and capable of hybridizing to at least a portion of another oligonucleotide or nucleic acid.
- Capture oligonucleotides can be immobilized to a solid support such that an oligonucleotide or nucleic acid that binds to the capture oligonucleotide becomes bound or tethered to the solid support.
- An oligonucleotide is said to be present in "excess" relative to another nucleic acid molecule if that oligonucleotide is present at a higher molar concentration than the other nucleic acid molecule.
- a primary cleavage oligonucleotide when a primary cleavage oligonucleotide is present in excess of a target nucleic acid it will be present in at least a 10 to 100-fold molar excess; typically at least 1 pmole of each primary cleavage oligonucleotide would be used when the target nucleic acid sequence is present at about 10 frnoles or less. See, for example, U.S. Patent No. 6,090,543.
- nucleic acid sequence refers to an oligonucleotide, nucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin which may be single or double-stranded, and represent the sense or antisense strand.
- FIG. 1 An overview of a system and method according to a preferred embodiment is shown in FIG. 1, and will now be described to illustrate the various components of the system.
- a target nucleic acid 10 is hybridized to a primary cleavage oligonucleotide 12.
- An invading oligonucleotide 14 also hybridizes to the target nucleic acid 10 to form a primary cleavage structure.
- a structure-specific nuclease cleaves the primary cleavage oligonucleotide 12, releasing a cleaved assay specific probe 16.
- the cleaved assay specific probe 16 hybridizes to a capture oligonucleotide 18 having a fluor 20 and a quencher 22.
- the capture oligonucleotide is coupled to a solid phase 24 in an array such that a specific location 26 in the array is associated with a specific target nucleic acid.
- a secondary cleavage structure is formed, and upon addition of nucleic acid cleavage means, the quencher 20 is released allowing the fluor to be capable of fluorescent resonance energy transfer (FRET).
- FRET fluorescent resonance energy transfer
- the solid substrate is then illuminated, generating emitted fluorescent light at any location where the quencher has been released.
- the resulting fluorescence is detected and the position in the array is mapped and conelated to known target nucleic acid positions to thereby identify the specific target nucleic acid present in the sample.
- Target Nucleic Acids The invention provides means for forming nucleic acid cleavage structures that are dependent upon the presence of a target nucleic acid.
- the nucleic acid cleavage structures are cleaved by a nucleic acid cleavage means to release distinct cleaved assay specific probes corresponding to each target nucleic acid.
- one or more target nucleic acids are derived from one or more samples. Each sample is known to contain, or is suspected of containing, one or more target nucleic acids.
- Target nucleic acids include various types of both RNA and DNA (including single stranded DNA and double stranded DNA) that are obtained according to standard techniques known in the art. For example, samples containing nucleic acids may be obtained from a tissue sample, tissue culture cells, samples containing bacteria and/or viruses, etc.
- the target nucleic acid may also be transcribed in vitro from a DNA template or may be synthetic, chemically synthesized, or generated via polymerase chain reaction (PCR). Further, nucleic acids may be isolated from organisms in the form of genomic material, plasmids, or similar extrachromosomal DNA, or the nucleic acid may be a fragment of such material generated by treatment with a restriction endonuclease or other cleavage agent. The binding of invading oligonucleotides 14 and primary cleavage oligonucleotides 12, described in more detail below, divides the target nucleic acid 10 into three distinct regions, as shown in FIG. 1.
- a first region 28 has complementarity to only the invading oligonucleotide 14; a second region 30, has complementarity to both the invading oligonucleotide 14 and the primary cleavage oligonucleotide 12; and a third region 32, has complementarity only to a portion of the corresponding primary cleavage oligonucleotide 12.
- the first region of each target nucleic acid 10 is contiguous to and downstream from the second region, and the second region of each target nucleic acid is contiguous to and downstream from the third region.
- a target nucleic acid may be either single-stranded or double-stranded.
- Double- stranded target nucleic acids can be rendered single stranded, for example by heating.
- the target nucleic acid can be reused or recycled during multiple rounds of hybridization with oligonucleotides and cleavage means.
- the methods of the invention are used to detect single nucleotide polymorphisms (SNP's). hi the examples described herein, the methods of the present invention used the Factor V (Leiden) SNP.
- Apolipoprotein E (ApoE)(C112R); Apolipoprotein E (ApoE)(R158C); Factor II (G320210A); Factor V (Leiden)(G1691A); Glycoprotein la (Gpla)(C807T); Glycoprotein Ilia (PL A1/A2)(T565C); Mehtylenetetrahydrofolate Reductase (MTHFR) (A1298C); Methlenetetrahydrofolate Reductase (MTHFR) (C677T) Plasminogen Activator Inhibitor-1 (PAL-1); ATP -Binding Cassette, Subfamily B Member 1; Cytochrome P450, Subfamily 1A, Polypeptide 1; Cytochrome P450 Subfamily 1A, Polypeptide 2; Cytochrome P450, Subfamily IIB, Polypeptide 6 Cytochrome P450, Subfamily
- each primary cleavage oligonucleotide comprises a target-specific (or non-flap) portion 34 and a cleavage (or flap) portion 36, located upstream of the target specific portion.
- the target-specific portion 34 of each primary cleavage oligonucleotide defines the third region 32 of the target nucleic acid sequence by being the complement of that region for a corresponding target nucleic acid.
- the cleavage portion 36 of each primary cleavage oligonucleotide defines the second region 30 of the target nucleic acid sequence by being the complement of that region for a corresponding target nucleic acid.
- the target nucleic acid strand that anneals with the primary cleavage oligonucleotide does so in a typical anti-parallel orientation.
- For each target nucleic acid there is a conesponding invading oligonucleotide 14.
- Each invading oligonucleotide comprises two portions, a cleavage portion and a target- specific portion.
- the cleavage portion of an invading oligonucleotide comprises a sequence complementary to the second region 30 of the corresponding target nucleic acid 10.
- the cleavage portion of an invading oligonucleotide 14 invades the cleavage portion 36 of the primary cleavage oligonucleotide.
- the invading oligonucleotide 14 is located upstream of the primary cleavage oligonucleotide 12 with respect to the target nucleic acid strand 10.
- Both the invading oligonucleotide 14 and the primary cleavage oligonucleotide 12 anneal to the corresponding target nucleic acid 10 in an anti-parallel orientation to the target nucleic acid strand.
- the target-specific portion of the invading oligonucleotide 14 comprises a sequence complementary to the first region 28 of the conesponding target nucleic acid 10.
- the invading oligonucleotide 14 and the primary cleavage oligonucleotide 12 are arranged in a parallel orientation relative to one another.
- the oligonucleotides of the invention are designed in accordance with methods that are known in the art. An initial consideration in choosing the length of oligonucleotides is the temperature under which they will be expected to be utilized for the methods described herein. For example, the chosen length of an oligonucleotide might vary depending on the thermal stability of the cleavage means.
- oligonucleotides are generally expected to have a higher hybridization specificity. It is desirable that the oligonucleotides of the invention have a length that is long enough to be reasonably expected to hybridize only to the intended target sequence within a complex sample.
- the oligonucleotide probes of the invention are typically 10 to 40 nucleotides in length, and more typically 25 to 35 nucleotides in length. See Lyamichev et al. (1999) Nature Biotechnology 17: 292-296; see also, U.S. Patent Nos. 6,562,611,5,994,069 and 5,846,717, the entire contents of which are hereby incorporated by reference.
- the length of the annealing portions are typically 10 to 40 nucleotides in length, and more typically 15 to 25 nucleotides in length. It is not intended that the method of the present invention be limited to any particular size of the primary cleavage oligonucleotide or invading oligonucleotide.
- the primary cleavage oligonucleotide can depart from the target nucleic acid after being cleaved, thus permitting the annealing and cleavage of other copies of the primary cleavage oligonucleotides ("turnover") without any discrete denaruration or displacement steps.
- One characteristic which is important in determining turnover is the melting temperature (Tm) of the primary cleavage oligonucleotide. This is effected both by length and G+C content of the oligonucleotide. In general, oligonucleotides with longer lengths and/or higher G+C content have higher melting temperatures.
- the cleavage (flap) portion of the primary cleavage oligonucleotide is released, leaving the target-specific (non-flap) portion annealed to the target nucleic acid. If the Tm of target-specific portion of the primary cleavage oligonucleotide is less than the reaction temperature, and the reaction temperature is less than the Tm of the entire primary cleavage oligonucleotide, then cleavage of the primary cleavage oligonucleotide will lead to the departure of target-specific portion of the primary cleavage oligonucleotide and a new primary cleavage oligonucleotide will be able to hybridize.
- the primary cleavage oligonucleotide is designed so that after cleavage of a portion of the primary cleavage oligonucleotide, the Tm of the remainder of the primary cleavage oligonucleotide is below the reaction temperature.
- Another factor affecting hybridization of the primary cleavage oligonucleotide is competition from the invading oligonucleotide.
- the portions of the primary cleavage and invading oligonucleotides that anneal with the first and third regions of the nucleic acid target are selected so as to have similar melting temperatures.
- the cleavage portion of the invading oligonucleotide may be favored in the competition for binding to the second region of the target.
- the invading oligonucleotide will still cause internal cleavage of the primary cleavage oligonucleotide, but the target-specific portion of the primary cleavage oligonucleotide bound to the third region of the target nucleic acid may not dissociate at the reaction temperature. This is likely to reduce the detection signal because the turnover would be less than optimal.
- Another objective in optimizing the cleavage assay is to allow specific detection of the fewest copies of a target nucleic acid.
- the combined elements of the reaction interact with the maximum efficiency, so that the rate of the reaction (e.g., the number of cleavage events per minute) is maximized.
- Elements contributing to the overall efficiency of the reaction include the rate of hybridization, the rate of cleavage, and the efficiency of the release of the cleaved probe.
- the rate of cleavage will be a function of the cleavage means chosen, and may be made optimal according to the manufacturer's instructions when using commercial preparations of enzymes or as described in the examples herein.
- the other elements depend upon the execution of the reaction.
- the concentration of the nucleic acids Three elements of the cleavage reaction that significantly affect the rate of nucleic acid hybridization are the concentration of the nucleic acids, the temperature at which the cleavage reaction is performed and the concentration of salts and/or other charge- shielding ions in the reaction solution. Methods for optimizing these conditions are well known in the art.
- the one or more samples comprising target nucleic acid, and in preferred embodiments more than one target nucleic acid, primary cleavage oligonucleotide(s), and invading oligonucleotide(s) are assembled into a buffered reaction mixture suitable for nucleic acid modification enzymes, the components of which are well known to those of skill in the art.
- the primary cleavage oligonucleotides are provided in sufficient excess (generally, at least a 100-fold molar excess) so that the rate of hybridization to the target nucleic acid(s) is rapid.
- the reactions are typically performed with at least 1 pmole of each primary cleavage oligonucleotide when the target nucleic acid sequence is present at about 10 fmoles or less.
- Other oligonucleotide concentrations, either higher or lower, commonly used in the art are contemplated and the methods described herein are not limited to these amounts.
- invading oligonucleotides it is advantageous for invading oligonucleotides to be immediately available to direct the cleavage of each primary cleavage oligonucleotide that hybridizes to a target nucleic acid.
- the primary cleavage oligonucleotide is provided in excess of the invading oligonucleotide in the reaction.
- An exemplary ratio of primary cleavage oligonucleotide to invading oligonucleotide are between a 2 and a 1000 fold excess of primary cleavage oligonucleotide over invading oligonucleotide.
- Nucleic Acid Cleavage Means A nucleic acid cleavage means encompasses any means that is capable of cleaving a cleavage structure, including but not limited to enzymes. Typically, the nucleic acid cleavage means comprise one or more "structure-specific nucleases". These structure- specific nucleases recognize specific secondary structures in a nucleic acid molecule and cleave these structures. The nucleic acid cleavage means includes native DNA polymerases having 5' nuclease activity (e.g., Taq DNA polymerase, E.
- coli DNA polymerase I nuclease activity provided from a variety of sources including the Cleavase enzymes, the FEN-1 endonucleases (including RAD2 and XPG proteins; AfuFEN, PfuFEN, MjaFEN, MthFEN), Taq and Tth DNA polymerases (including TaqPol, TaqExo, and TthPol) and E. coli DNA polymerase I.
- the cleavage means includes DNA polymerases that have been modified so as to have a 5' nuclease without synthetic activity.
- the nucleic acid cleavage means further encompasses 5' nucleases derived from thermostable Type A DNA polymerases that retain 5' nuclease activity but have reduced or absent synthetic activity. Modified enzymes having reduced synthetic activity and increased 5' nuclease activity relative to unmodified enzymes are also envisioned by the present invention. Typically, the modified enzyme may have no synthetic activity remaining or may have that level of synthetic activity that will not interfere with the use of the modified enzyme in the detection assay described below. Exemplary nucleic acid cleavage means are known in the art, and are described in more detail in Kaiser et al. (1999) J. Biol. Chem. 274: 21387-21394; U.S. Patent No.
- cleavage structure refers to a structure that is formed by the interaction of one or more oligonucleotides and a target nucleic acid to form a duplex, where the resulting structure is cleavable by a cleavage means.
- the cleavage structure is a substrate for specific cleavage by the cleavage means, as opposed to a nucleic acid molecule that is a substrate for non-specific cleavage by agents such as phosphodiesterases that cleave nucleic acid molecules without regard to secondary structure (i.e., no formation of a duplexed structure is required).
- the products generated by the reaction of a cleavage means with a cleavage structure are referred to herein as "cleavage products”.
- a primary cleavage structure is generated for each target nucleic acid by the interaction of each target nucleic acid with the corresponding primary cleavage oligonucleotide and the corresponding invading oligonucleotide.
- each primary cleavage structure at least the 3' portion of each primary cleavage oligonucleotide is annealed to a corresponding target nucleic acid. As described above, the 3' portion of the primary cleavage oligonucleotide anneals to the third region of the corresponding target nucleic acid. At least the 5' portion of each invading oligonucleotide is annealed to a first region of the corresponding target nucleic acid.
- the primary cleavage structures are cleaved by the nucleic acid cleavage means to generate a cleavage product termed herein as a "cleaved assay specific probe".
- cleaved assay specific probe formed conesponding to each target nucleic acid.
- Each cleaved assay specific probe comprises a 5' region and a 3' region.
- Secondary Cleavage In one embodiment of the instant invention, a secondary cleavage reaction is performed where the cleavage product of the first stage interacts with a unique capture oligonucleotide for each target and forms a secondary cleavage structure. Subsequent sequential cleavage reactions are within the scope of the invention, including tertiary cleavage reactions and so forth. The cleavage of the secondary cleavage structure or formation of second stage cleavage products can be used as a basis of detection or to drive further stages of sequential cleavage reactions.
- the product of the secondary cleavage reaction is not capable of initiating either of the first or second stage cleavage reactions.
- the reaction components for the first stage cleavage are mixed with components for the second stage cleavage reaction so that second stage reactions can be initiated directly after product from the primary cleavage reaction becomes available.
- the primary and secondary cleavage events can take place simultaneously.
- the reaction format can also be configured with each step of cleavage reactions being spatially or temporally separated, such as performing each stage in different reaction vessels or by causing a change in reaction conditions that allow later cleavage events to take place.
- each capture oligonucleotide 18 comprises contiguous first and second regions.
- the first region 38 comprises the 3' end of the capture oligonucleotide.
- the second region 40 comprises the 5' end of the capture oligonucleotide.
- the second region of the capture oligonucleotide further comprises a region of self-complementarity that is capable of forming a hairpin loop.
- the first region of each capture oligonucleotide is complementary to a corresponding cleaved assay specific probe that is generated in the primary cleavage reaction.
- a portion of the second region of each capture oligonucleotide is complementary to at least a portion of the 3' region of the corresponding cleaved assay specific probe.
- capture oligonucleotides are deposited onto a solid substrate, with the location of each type of capture oligonucleotide mapped to a known position on the surface of the substrate.
- FIG. 2 One type of solid substrate useful in the practice of the present invention is shown in FIG. 2.
- capture oligonucleotides are deposited in a 4 x 4 array 42 in each well 44 of an activated microplate, such as an activated polypropylene plate 46.
- an activated microplate such as an activated polypropylene plate 46.
- wells are arranged in rows 48 and columns 50.
- 5'-arnino terminated capture oligonucleotides can be immobilized in the bottom of the wells of an acyl fluoride (ACF) activated polypropylene microplate using acyl fluoride surface activation, as described below in Example 3.
- ACF acyl fluoride
- Other means for attaching amino- oligonucleotide capture probes are known in the art, and may be used with the present invention. These include, but are not limited to, the use of epoxide, aldehyde or imidazole chemistries. Other examples are shown in U.S. Patent No. 6,146,833, the content of which is hereby incorporated by reference in its entirety..
- each capture oligonucleotide deposited at each predetermined site on the solid substrate is typically in the femtomole to picomoles range per reaction. Other oligonucleotide concentrations, either higher or lower, commonly used in the art are contemplated and the methods described herein are not limited to these amounts.
- each capture oligonucleotide typically comprises a detectable label or moiety, generally placed at or near the 5' end of the secondary cleavage oligonucleotide, or may be positioned anywhere along the oligonucleotide.
- the detectable label can be dye such as a fluorophore.
- the capture oligonucleotides comprise a fluorescent donor and fluorescent acceptor that generate a fluorescence-based signal in response to a change in distance between the fluorescent donor and acceptor that is caused by cleavage.
- the 5' end of each capture oligonucleotide comprises one or more detectable labels.
- the 5' end of each capture oligonucleotide comprises a fluorescent donor and fluorescent acceptor that generate a fluorescence-based signal in response to cleavage of the secondary cleavage structure.
- the fluorescent analyte detection dye can be a single fluorescer or a donor-receptor dye pair that is activated by energy transfer in the detection system and can be synthetic or a naturally occurring fluorescer.
- Appropriate fluorescent analyte detection dyes can be selected for a particular assay and used in accordance with the present invention by those of skill in the art with reference to this disclosure.
- Known fluorescent dyes useful for labeling oligonucleotides include hydrophobic and stable dyes such as those known by the designations IR792 ([2-[2-[3-[(l,3-dihydro- 3,3-dimethyl-l-propyl-2H-indol-2-ylidine)ethylidine]-2-(phenylthio)-l-cyclohexen-l- yl]ethenyl]-3,3-dimethyl-l-pro ⁇ ylindolium perchlorate]), IR768 ([2-[2-[3-[(l,3-dihydro- 3 , 3 -dimethyl- 1 -propyl-2H-indol-2-ylidine)ethylidine]-2-phenoxy- 1 -cyclohexen- 1 - yl]ethenyl]-3,3-dimethyl-l-propylindolium perchlorate])YL22 ([2-[2-[
- Prefened dye structures that can be associated with capture oligonucleotides include IR792, YL22, and the lipophilic forms of cyanine dyes known under the designations Cy7 (e.g. DiIC18 (7) from Molecular Probes, Inc.) and dibenzoCy7, Cy5, and dibenzoCy5.
- Cy7 e.g. DiIC18 (7) from Molecular Probes, Inc.
- dibenzoCy7 e.g. DiIC18 (7) from Molecular Probes, Inc.
- Cy5 e.g. DiIC18 (7) from Molecular Probes, Inc.
- Cy5 e.g. DiIC18 (7) from Molecular Probes, Inc.
- dibenzoCy7 e.g. DiIC18 (7) from Molecular Probes, Inc.
- Cy5 e.g. DiIC18 (7) from Molecular Probes, Inc.
- dibenzoCy7 e.g. DiIC18 (7) from Molecular Probes, Inc.
- Cy5
- Fluorescing cyanine dyes are commercially available.
- Light energy selected so as to excite the fluorescent label used in the assay is used to illuminate the capture oligonuleotides bound to the solid substrate, to thereby generate a fluorescent signal.
- a light source such as a laser or an arc lamp may be used.
- an excitation laser is used to excite the fluorescent labels.
- Appropriate laser line filters, beam expanders, minors, and lenses may also be used, as will be understood by those with skill in the art with reference to this disclosure.
- a prefened excitation wavelength for the fluorescent analyte detection dyes is a 532 nm diode laser, alternatively, a 635 nm diode laser, a 650 nm diode laser, or a 633 nm helium-neon laser can be used.
- a lower wavelength 488 nm argon-ion, or a 532 nm doubled YAG laser can be used.
- Longer wavelength lasers e.g., greater than 750 nm
- a prefened longer laser excitation wavelength is about 785 nm.
- detectors for detecting a particular emitting light are known as will be understood by those with skill in the art with reference to this disclosure.
- the detectors can be photodiodes or photomultipliers or similar devices that convert light signals into electrical impulses thereby associating the detected light with its fluorescent source.
- the detection subsystem can also employ a system of filters, minors, as will be understood by those with skill in the art with reference to this disclosure.
- the electrical signals from the detectors are typically fed into the electronics of the system for signal and display processing, storage, and/or further processing.
- light signals may be detected and recorded using a charge coupled device (“CCD”) camera, as will be appreciated by one of skill in the art.
- CCD charge coupled device
- Hardware such as a microprocessor in combination with memory storage such as a hard drive in a computer, may be used to collect detected data and to process the data.
- Suitable hardware used in the analysis system is known as will be understood by those with skill in the art with reference to this disclosure.
- the analysis system software used for data and signal processing, can conelate detected data with known data to produce analytical results.
- the analysis subsystem can collect data from the electrical signals associated with each specific location on the solid substrate.
- the processed data and interpreted results can be given as output to a user.
- a method of using solid substrate arrays to detect target nucleic acids is shown in FIG. 3.
- a solid substrate having a set of capture nucleotides specific for target nucleic acids deposited on the solid surface in a previously determined array is selected, box 52.
- the capture oligonucleotides are then exposed to the sample and necessary reagents, box 54. Following exposure, the capture oligonucleotides, and any cleaved assay specific probe hybridized thereto, are processed in an examination zone.
- the capture oligonucleotides are processed by illuminating the capture oligonucleotides, generating emitted fluorescent light and detecting the emitted light, box 56.
- the detected emitted light data is correlated with the baseline data to determine the detection of one or more target nucleic acids, box 58.
- an analytical sample is allowed to react with a set of oligonucleotides, which are specific to various analytes of interest and which are bound in a predetermined anangement to a solid substrate.
- the solid substrate is then illuminated such that capture oligonucleotides that have reacted with their specific analyte of interest generate a fluorescent signal.
- a detection device identifies the location of the emitted signal and conelates this information with the information about the location of the analyte specific capture oligonucleotide to allow quantitative identification of multiple analytes in one reaction.
- the method of the present invention is used to detect single nucleotide polymorphisms (SNPs) in nucleic acids, as shown in FIG. 4.
- the target of interest in the target nucleic acid 10 is a specific SNP 60; in this example, the SNP contains a thymine (T).
- T thymine
- a set 62 of four different types of primary cleavage oligonucleotides are added to the target nucleic acid.
- All of these primary cleavage oligonucleotides contain a 3' portion 34, which is at least partially complementary to the target nucleic acid immediately adjacent to and upstream of the site of the SNP, and which terminates at the 5' end with one of four nucleotides, containing either adenine (A, 64), cytosine (C, 66), guanine (G, 68) or thymine (T, 70).
- each primary cleavage oligonucleotide contains a 5' portion containing a SNP bar code sequence (65, 67, 69, 71) unique for the particular SNP.
- the primary cleavage oligonucleotide having an A has a bar code sequence 65 (the "A" bar code sequence) which serves to distinguish it from the other primary cleavage oligonucleotides.
- the 3' portion 34 of the primary cleavage oligonucleotide hybridizes with the target nucleic acid, only one of the four sets of primary cleavage oligonucleotides, in this case the set containing an A, will also hybridize at the site of the SNP to form a primary cleavage structure 72 upon addition of invading oligonucleotide 14.
- the set containing an A will also hybridize at the site of the SNP to form a primary cleavage structure 72 upon addition of invading oligonucleotide 14.
- the resulting cleaved assay specific probe 16 will contain an A at its 3' end.
- Each capture oligonucleotide also contains the complement to an SNP bar code sequence in its 3' portion; for example, a capture oligonucleotide containing a T between the fluor and the quencher (a "T" capture oligonucleotide) is designed so as to have sequence complementary to the "A" SNP bar code in its 3' portion.
- the capture oligonucleotides are exposed to the cleaved assay specific probe, which hybridizes to the capture oligonucleotides.
- the cleaved assay specific probe contains an A at its 3' end and an "A" bar code sequence in its 5' end.
- the quencher is cleaved off the secondary cleavage structure, resulting in the emission of fluorescent light.
- the fluorescent light is detected and its position conelated with the predetermined map locations of the capture oligonucleotides to thereby identify the SNP in the target nucleic acid.
- the fluor remains covalently bound to the capture oligonucleotide, and thus to the solid support, following cleavage of the secondary cleavage structure.
- this maintains the fluorescent signal at the predetermined map location in an array to facilitate detection and correlation of the signal to a specific location.
- the position of the fluor and the quencher on the capture oligonucleotide may be reversed, as shown in FIG. 4 (78).
- the primary cleavage reaction is performed in a separate reaction mix so as to allow accumulation of the cleaved assay specific probe.
- This reaction is then terminated, as by denaturing the nucleic acid cleavage means, and the contents of the reaction transfened to the immobilized capture oligonucleotides.
- the fluor is released into solution.
- multiple genetic loci can be screened for SNPs in multiplexed assays using multiple bar codes, as shown in FIGs. 5 and 6.
- primary cleavage oligonucleotides are designed to include both an SNP bar code and a locus bar code.
- two first cleavage nucleotides 80, 82 are shown, each of which contains the SNP bar code for A (84).
- one first cleavage nucleotide 80 contains a loci bar code (86) for loci 1, while the other 82 contains a loci bar code (88) for loci 2.
- these two primary cleavage oligonucleotides 80, 82 may be used in the present invention to identify "A" SNPs at gene loci 1 and 2, respectively.
- two primary cleavage oligonucleotides 90, 92 are shown for the same gene loci, in this case, loci 1.
- Each oligonucleotide contains the loci 1 bar code 86, however, one primary cleavage oligonucleotide contains an SNP bar code for G (94) while the other contains an SNP bar code for T (96).
- these two primary cleavage oligonucleotides 90, 92 may be used in the present invention to identify a "G" SNP or a "T" SNP, respectively, at gene loci 1.
- each gene loci of interest requires four primary cleavage oligonucleotides, each with the appropriate SNP bar code, for the four nucleotides potentially found at the site of the polymorphism, i.e., A, C, G, and T.
- unique primary cleavage oligonucleotides can be designed so that the entire sequence of the 5' portion of the oligonucleotide serves as an indicator of both SNP and gene locus.
- each locus would require four unique primary cleavage oligonucleotides conesponding to the SNP identity (A, C, G, T) and the locus identity.
- four hundred unique hairpin capture oligonucleotides would be needed to map one hundred SNPs by perfect match hybridization to the four hundred unique cleaved assay specific probes.
- cleavage oligonucleotides are labeled with biotin and cleavage products are detected by ELF signal amplification following hybridization with capture oligonucleotides affixed to a solid substrate, as shown in FIG. 7.
- a set 83 of four unique primary cleavage oligonucleotides are provided for each loci of interest in the target nucleic acid 10, each of which is designed to include both an SNP barcode and a loci barcode in the 3' portion, as described above.
- each primary cleavage oligonucleotide is labeled with biotin 100.
- the primary cleavage oligonucleotides are hybridized to target nucleic acid in the presence of invading oligonucleotides and nucleic acid cleavage means as described above, and following termination of the cleavage reaction, as by, for example, denaturation of the nucleic acid cleavage means, the resulting cleaved assay specific probe 102 is recovered.
- Capture oligonucleotides are designed so as to be complementary to the various cleaved assay specific probes that may be generated in the primary cleavage reaction.
- capture oligonucleotides are deposited onto a solid substrate in predetermined locations as described above.
- each SNP at each locus of interest may be mapped to a specific site in the array.
- Cleaved assay specific probe from the cleavage reaction is transfened to the solid substrate and allowed to hybridize to capture oligonucleotides containing complementary sequence. Since each locus and SNP may be identified by its specific location in the array, only a single reporter label is required.
- Enzyme-Labeled Fluorescence (ELF) signal amplification may be used to detect hybridization to capture oligonucleotides via CCD camera imaging and digital quantitation, and thus to identify the target nucleic acid of interest, as shown in FIG. 8.
- ELF Enzyme-Labeled Fluorescence
- Other signal detection systems may also be used, including detection by chemiluminescence.
- other labels than biotin may be employed, including fluorescein isothiocyanate (FITC) or Cy- series near infrared dyes such as Cy3 or Cy5.
- this embodiment allows for high-throughput screening while eliminating the need for hairpin capture oligonucleotides, labeled capture oligonucleotides, or multiple labels for multiple targets.
- Dual-Labeled Probe it may be desirable to remove uncleaved primary cleavage oligonucleotide prior to exposure to capture oligonucleotides.
- a method is provided wherein dual-labeled primary cleavage oligonucleotides are used.
- a detectable label as for example, FITC
- an affinity tag such as biotin
- the assay mix is contacted with a compound having affinity for the affinity tag, such as sfreptavidin in the case of biotin.
- sfreptavidin coated beads may be used to sequester any fragments containing biotin.
- sfreptavidin coated beads are used in a device such as the bio-tip described in U.S. Patent No. 5,437,979.
- magnets may be used to remove sfreptavidin coated magnetic or paramagnetic beads.
- cleaved assay specific probes containing only the 5' label may be used for hybridization with capture oligonucleotides without competition from uncleaved reactants.
- Dual-labeled primary cleavage oligonucleotides are also useful for monitoring the specific activity of nucleic acid cleavage means, such as Cleavase enzymes, as shown in FIG. 9.
- the primary cleavage oligonucleotide is labeled at each end using distinguishable reporters.
- Two sets of capture oligonucleotides are designed, one set 110 complementary to the 5' end of the primary cleavage oligonucleotide (FLAP) 112, conesponding to the cleaved assay specific probe, and the other set 114 complementary to the 3' end 116. Following the cleavage reaction, reactants are contacted to the capture oligonucleotides and the signal intensities of the captured oligonucleotides are determined.
- FLAP primary cleavage oligonucleotide
- Enzyme specific activity may be determined using the ratio of 5'-label only (conesponding to the cleaved assay specific probe 118) to 5'-label plus 3' label (conesponding to uncleaved primary cleavage oligonucleotide 120). This allows for an estimation of initial velocity and end-points of the reaction.
- the primary cleavage oligonucleotide can be internally labeled.
- Reagent Sets The present invention also contemplates kits and reagent sets useful for practice of the methods described herein.
- kits or reagent sets may include, for example, primary cleavage oligonucleotides, invading oligonucleotides, and capture oligonucleotides deposited onto solid substrates specific for target nucleic acids of interest, control target nucleic acids, nucleic acid cleavage means, various labels and detection means, and instructions for practice of the invention.
- Example 1 Cleavage Reaction A reaction mixture was prepared as follows: Invading oligonucleotide (1 ⁇ L of 0.5 ⁇ M stock solution) was mixed with 4 ⁇ L of PEG (16% v/v in 50 mM MOPS, pH 7.5) and the solution placed in a 0.2 L Eppendorf tube. Next, 10 ⁇ L of DNA sample target (50 attomoles) was added per tube and overlayed with 20 ⁇ L of mineral oil. The invading oligonucleotide-target mixture was incubated for 5 minutes at 95°C to denature double stranded targets.
- the temperature was reduced to 59°C and 5 ⁇ L of Cleavase enzyme (Third Wave) mixture (2 ⁇ L, 75mM MgCl 2 + 1 ⁇ L Cleavase, 100 ng + 1 ⁇ L dual-labeled (5'-FITC, 3'-biotin) primary cleavage oligonucleotide, 377 nM + 1 ⁇ L distilled water) added to the tube.
- the reaction was allowed to proceed 4 hours at 59°C.
- Example 2 Cleavage Product Processing
- the reaction mixture from Example 1 was cooled to 25° C and processed to remove biotinylated primary cleavage oligonucleotide fragment and residual cleavage oligonucleotide from the FITC-labeled cleaved assay specific probe.
- the reaction mixture (20 ⁇ L) was mixed with 30 ⁇ L of streptavidin-coated magnetic beads (Calbiochem) and the solution vortexed. The solution was maintained for 1 hour using an end-over-end rotator. Beads were removed from solution by placing the tube next to a magnetic strip for 1-2 minutes.
- Example 3 Capture Oligonucleotide Plate Preparation
- the 5 '-amino terminated capture oligonucleotides complementary to the cleaved assay specific probe were immobilized in the bottom of the wells of an acyl fluoride (ACF) activated polypropylene microplate.
- ACF acyl fluoride
- 10 nL of 10 ⁇ M amino capture oligonucleotide prepared in 50 mM sodium carbonate buffer, pH 9, containing 50% v/v MSS (MicroSpotting Solution, Telechem) was deposited on the bottom of the well by contact printing.
- a Biomek 2000 equipped with an HDRT pin system (Beckman Coulter) was used to deliver the oligonucleotide droplets to the well surface.
- the wells were incubated with reagent grade ammonium hydroxide (30% v/v water) for 15 minutes to remove unbound oligonucleotide and cap residual acyl fluoride groups. The wells were then rinsed with water, dried and stored sealed under argon at -20 °C until required for the hybridization experiments.
- Example 4 Solid-Phase Hybridization
- the supernatant containing the cleaved assay specific probes (50 ⁇ L) from Example 2 was mixed with 30 ⁇ L of SSC Hybridization Buffer containing SDS to bring the final concentration of hybridization buffer to 6X SSC, 0.01% SDS.
- the 80 ⁇ L hybridization mixture was applied to the well of the hybridization plate containing complementary capture oligonucleotides immobilized to the bottom of the well from Example 3.
- the hybridization was allowed to proceed for 1 hour at 25° C in a humidified shaker-water bath.
- the solution was removed from the wells and each well rinsed 3 -times with 2X SSC, 0.02%) Tween.
- An anti-FITC antibody-alkaline phosphatase conjugate (Sigma- Aldrich) was prepared in rinse buffer at 1:1000 v/v and 50 ⁇ L applied to each well. The conjugate was incubated for 1 hour at 25° C in a humidified shaker-water bath and processed as previously described. ELF reagent (Molecular Probes) was prepared at 1:100 v/v and 50 ⁇ L applied to each well for 30 minutes. After a brief buffer (SSC- Tween) rinse, the signal was detected using a CCD camera with fluorescent light excitation at 365 nm with lens filtering at emission 520nm. Examples 1-4 are summarized schematically in FIG. 10. In FIG.
- a primary cleavage structure is formed from target nucleic acid (130) with invading oligonucleotide (132) and primary cleavage oligonucleotide (134).
- the primary cleavage oligonucleotide is dual-labeled with 5' FITC and 3' biotin.
- the action of the nucleic acid Cleavase enzyme (136) results in cleavage of the primary cleavage oligonucleotide (134) into the cleaved assay specific fragment (138) and the cleaved primary cleavage oligonucleotide fragment (140).
- the biotinylated primary cleavage oligonucleotide fragment (140) and remaining primary cleavage oligonucleotide (134) are captured by streptavidin- immobilized magnetic beads (142) which are in turn sequestered or removed from the solution by magnet (144).
- the FITC-labeled cleaved assay specific fragment (138) is hybridized from solution using the complementary capture oligonucleotide (146) which is immobilized onto solid-support (148). After appropriate rinsing, an anti-FITC antibody- reporter enzyme conjugate (150) is added to the solid-support (148) and signal developed.
- Example 5 Effect of Varying the Concentration of Primary Cleavage Oligonucleotide on Removal of Uncleaved Primary Cleavage Oligonucleotide.
- Primary cleavage structure was prepared with variation in the amount of dual- labeled primary cleavage oligonucleotide (from 37.7 to 377 finoles) and allowed to undergo cleavase treatment for 4 hours at 59 °C as described in Example 1. The temperature was reduced to 25 °C and the reaction mixture incubated with streptavidin- immobilized magnetic beads for 1 hour at 25 °C. After removal of the beads, the supernatant was prepared in hybridization buffer and incubated in wells of the oligonucleotide capture array plate for an additional 1 hour.
- Example 6 Dual Labeled Primary Cleavage Oligonucleotide.
- the assay described above in Examples 1-4 was performed using a dual labeled primary cleavage oligonucleotide, and the effectiveness of scavenging uncleaved product assessed.
- Biotin-label detection was performed using sfreptavidin alkaline phosphatase.
- FITC-label detection was determined using an anti-FITC alkaline phosphatase conjugate. Both signals were developed using the ELF reagent with CCD camera imaging and digital quantitation. The results are shown in FIG. 12.
- (A,B) the wild type primary cleavage oligonucleotides were labeled with ST-uotin.
- reaction mixture was either used directly for hybridization to the anti-cleaved assay specific probe capture oligonucleotide (A) or first exposed to sfreptavidin coated magnetic beads in order to sequester 5' biotin labeled oligonucleotides (B).
- A substantial capture of both the full (uncleaved) primary cleavage oligonucleotide and the cleaved assay specific probe are indicated, while in B the biotin-labeled probes are efficiently captured by beads resulting in no capture by anti-cleaved assay specific probe capture oligonucleotides.
- FDLPB dual-labeled primary cleavage oligonucleotide
- the dual labeled FDLPB is effectively scavenged by magnetic bead capture, while the cleavage product (5' FITC labeled cleaved assay specific probe) is quantitatively captured by the anti- cleaved assay specific probe capture probe.
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Abstract
An assay method and reagents useful for detecting and characterizing multiple specific target nucleic acids and/or sequence variations in a single multiplexed reaction is provided.
Description
SOLID-PHASE MULTIPLEXED INVADER ASSAY
BACKGROUND Assay methods have been described which are useful for the detection and characterization of specific target nucleic acids and sequence variations within such specific nucleic acids, including single nucleotide polymorphisms or SNPs. One such assay has been described, which utilizes nucleic acid cleavage structures that are cleaved in a site-specific manner to indicate the presence of specific nucleic acid sequences. See Brow, et al, U.S. Patent No. 5,846,717 and Hall, et al., U.S. Patent No. 5,994,069. Such methods have great utility in the detection and diagnosis of various genetic diseases and disorders, in the identification of gene mutations, and in many other contexts, as will be appreciated by the skilled artisan. Typically, however, existing approaches do not readily lend themselves to simultaneous detection of multiple targets. Although some multiplexed assays have been developed using, for example, multiple fluorescent labels, such methods have a number of limitations, including increased cost and a limit on the number of suitable labels that can be used simultaneously in multiplexed assays. What is needed, therefore, is an assay method that can detect and characterize multiple specific target nucleic acids and/or sequence variations in a single reaction (a "multiplexed" assay) that is efficient, reliable, and cost effective.
SUMMARY OF THE INVENTION In one embodiment, the invention includes a method for detecting the presence of a target nucleic acid comprising: (a) providing: (i) a sample comprising at least one target nucleic acid having a first portion, a second portion, and a third portion, wherein the first portion is downstream of and contiguous to the second portion, the second portion is downstream of and contiguous to the third portion, and wherein each target nucleic acid is associated with a preselected bar code sequence; (ii) an invading oligonucleotide, having a 3' portion and a 5' portion, the 5' portion containing sequence complementary to the first portion of the target nucleic acid and the 3' portion containing sequence complementary to the second portion of the target nucleic acid; (iii) a primary cleavage oligonucleotide, each primary cleavage oligonucleotide having a 3' portion complementary to the third portion of the target nucleic acid, a cleavage portion complementary to the second portion of the target nucleic acid, and a 5' portion having a bar code sequence specific for the target nucleic acid; (iv) a nucleic acid cleavage means; (b) generating a primary cleavage structure for each target nucleic acid, wherein at least the 3' portion of the primary cleavage oligonucleotide is annealed to a corresponding target nucleic acid and at least the
5' portion of the invading oligonucleotide is annealed to a corresponding target nucleic acid, wherein cleavage of the primary cleavage structure is performed by the nucleic acid cleavage means to cleave each primary cleavage oligonucleotide to generate a cleaved assay specific probe corresponding to each target nucleic acid, each cleaved assay specific probe having a 5' region and a 3' region; (c) providing a capture oligonucleotide for each target nucleic acid, each capture oligonucleotide having contiguous first and second portions, wherein the first portion comprises a sequence complementary to the bar code sequence associated with the target nucleic acid and a 3' end of the capture oligonucleotide, the 3' end of the capture oligonucleotide is coupled to a solid support in a predetermined location, such that each capture oligonucleotide bar code sequence is associated with a specific site on the solid support, wherein the second portion comprises a 5' end of the capture oligonucleotide, a region of self-complementarity, and is coupled to a detectable label in a first state, wherein the first portion of each capture oligonucleotide is complementary to a cleaved assay specific probe for a corresponding target nucleic acid, and wherein a part of the second portion of each capture oligonucleotide is complementary to at least a portion of the 3' region of the corresponding cleaved assay specific probe; (d) generating a secondary cleavage structure, wherein the 5' end of each capture oligonucleotide is cleaved from the remainder of the second region of each capture oligonucleotide changing the detectable label to a second state; (e) detecting the cleavage of the capture oligonucleotides by a change in fluorescence; and (f) associating the detected change in fluorescence with a specific site on the solid substrate thereby identifying the bar code sequence associated with the change in fluorescence and detecting the target nucleic acid corresponding to the bar code sequence. hi another embodiment, the invention includes a method for detecting and identifying a single nucleotide polymorphism (SNP) in a target nucleic acid molecule in an assay, comprising: (a) providing: (i) at least one target nucleic acid molecule having a first portion, a second portion comprising a SNP, and a third portion, wherein the first portion is downstream of and continguous to the second portion, the second portion is downstream of and contiguous to the third portion; (ii) an invading oligonucleotide, having a 3' portion and a 5' portion, the 5' portion of the invading oligonucleotide containing sequence complementary to the first portion of the target nucleic; (iii) a set of primary cleavage oligonucleotides, each primary cleavage oligonucleotide having a 3' portion complementary to the third portion of the target nucleic acid, a middle portion containing a nucleotide having a base selected from the group consisting of A, T, C, and G, and a 5' portion containing a SNP-specific bar code sequence; (iv) a nucleic acid cleavage means; (b) mixing the target nucleic acid, the invading oligonucleotides, the set
of primary cleavage oligonucleotides and the nucleic acid cleavage means to create a reaction mixture under reaction conditions such that at least the 3' portion and middle portion of those primary cleavage oligonucleotides having a middle portion complementary to the SNP portion of the target nucleic acid are annealed to the target nucleic acid and wherein at least the 5' portion of the invading oligonucleotide is annealed to the target nucleic acid so as to create a primary cleavage structure, wherein cleavage of the primary cleavage structure occurs to generate a cleaved assay specific probe comprising the 5' portion and middle portion of those primary cleavage oligonucleotides having a middle portion complementary to the SNP portion of the target nucleic acid, such that the cleaved assay specific probe terminates at its 3' end with a nucleotide complementary to the SNP portion of the target nucleic acid; (c) providing an array comprising a set of capture oligonucleotides and a solid phase, each capture oligonucleotide having a 5' portion, a middle portion, and a 3' portion, the 3' portion being attached at the 3' end to the solid phase and containing sequence complementary the SNP- specific bar code sequence, the middle portion having a sequence capable of forming a hairpin loop, the middle portion being labeled with a reporter and terminating at its 5' end with a nucleotide containing a base selected from the group consisting of A, C, T, and G, and the 5' portion containing a quencher, such that the quencher interacts with the reporter to prevent the reporter from producing a signal, wherein the bar code sequence of each capture oligonucleotide is associated with a location on the array; (d) adding the cleaved assay specific probe from the reaction mixture to the array to create an array mixture under reaction conditions such that at least the 5' portion of the cleaved assay specific probe is annealed to the 3' portion of the capture oligonucleotide and the nucleotide at the 3' end of the cleaved assay specific probe is annealed to the nucleotide at the 5' end of the middle portion of the capture oligonucleotide so as to create a secondary cleavage structure; (e) adding nucleic acid cleavage means to the array mixture to thereby cleave the second cleavage structure, releasing the quencher from the capture oligonucleotide; (f) detecting signal from the reporter and associating it with its location on the array to identify the bar code sequence contained in the capture oligonucleotides from which the quencher was removed to thereby identify the SNP in the target nucleic acid. hi another embodiment, the invention includes a method for detecting and identifying a single nucleotide polymorphism at a specific gene locus in a target nucleic acid molecule in an assay, comprising: (a) providing: (i) at least one target nucleic acid molecule having a first portion, a second portion comprising a SNP, and a third portion, wherein the first portion is downstream of and continguous to the second portion, the second portion is downstream of and contiguous to the third portion; (ii) an invading oligonucleotide, having a 3' portion and a 5' portion, the 5' portion of the invading
oligonucleotide containing sequence complementary to the first portion of the target nucleic; (iii) at least one set of primary cleavage oligonucleotides for each gene locus, each primary cleavage oligonucleotide having a 3' portion complementary to the third portion of the target nucleic acid, a middle portion containing a nucleotide having a base selected from the group consisting of A, T, C, and G, and a 51 portion containing an SNP- specific bar code sequence and a gene locus-specific bar code sequence; (iv) a nucleic acid cleavage means; (b) mixing the target nucleic acid, the invading oligonucleotides, the sets of primary cleavage oligonucleotides and the nucleic acid cleavage means to create a reaction mixture under reaction conditions such that at least the 3' portion and middle portion of those primary cleavage oligonucleotides having a middle portion complementary to the SNP portion of the target nucleic acid and a 3' portion complementary to the gene locus on the target nucleic acid are annealed to the target nucleic acid and wherein at least the 5' portion of the invading oligonucleotide is annealed to the target nucleic acid so as to create a primary cleavage structure, wherein cleavage of the primary cleavage structure occurs to generate a cleaved assay specific probe comprising the 5' portion and middle portion of those primary cleavage oligonucleotides having a middle portion complementary to the SNP portion of the target nucleic acid, such that the cleaved assay specific probe terminates at its 3' end with a nucleotide complementary to the SNP portion of the target nucleic acid; (c) providing an array comprising a set of capture oligonucleotides and a solid phase, each capture oligonucleotide having a 5' portion, a middle portion, and a 3' portion, the 3' portion being attached at the 3' end to the solid phase and containing sequence complementary the SNP- specific bar code sequence and the gene locus bar code sequence, the middle portion having a sequence capable of forming a hairpin loop, the middle portion being labeled with a reporter and terminating at its 5' end with a nucleotide containing a base selected from the group consisting of A, C, T, and G, and the 5' portion containing a quencher, such that the quencher interacts with the reporter to prevent the reporter from producing a signal, wherein the bar code sequences of each capture oligonucleotide are associated with a location on the array; (d) adding the cleaved assay specific probe from the reaction mixture to the array to create an array mixture under reaction conditions such that at least the 5' portion of the cleaved assay specific probe is annealed to the 3' portion of the capture oligonucleotide and the nucleotide at the 3' end of the cleaved assay specific probe is annealed to the nucleotide at the 5' end of the middle portion of the capture oligonucleotide so as to create a secondary cleavage structure; (e) adding nucleic acid cleavage means to the array mixture to thereby cleave the second cleavage structure, releasing the quencher from the capture oligonucleotide; (f) detecting signal from the reporter and associating it with its location on the array to identify the bar code sequences
contained in the capture oligonucleotides from which the quencher was removed to thereby identify the SNP and gene locus in the target nucleic acid. In another embodiment, the invention includes a method of detecting and identifying a single nucleotide polymorphism (SNP) in a target nucleic acid molecule in an assay, comprising: providing: (i) a target nucleic acid molecule having a first portion, a second portion comprising a SNP, and a third portion, wherein the first portion is downstream of and contiguous to the second portion, the second portion is downstream of and contiguous to the third portion; (ii) an invading oligonucleotide, having a 3' portion and a 5' portion, the 5' portion of the invading oligonucleotide containing sequence complementary to the first portion of the target nucleic; (iii) a set of primary cleavage oligonucleotides, each primary cleavage oligonucleotide having a 3' portion complementary to the third portion of the target nucleic acid, a middle portion containing a nucleotide containing a base selected from the group consisting of A, T, C, and G, and a 5" portion containing an SNP-specific bar code sequence; (iv) an array comprising a set of capture oligonucleotides and a solid phase, each capture oligonucleotide having a 5' portion, a middle portion, and a 3' portion, the 3' portion being attached at the 3' end to the solid phase and containing sequence complementary the SNP-specific bar code sequence, wherein the bar code sequence of each capture oligonucleotide is associated with a location on the array, the middle portion having a sequence capable of forming a hairpin loop, the middle portion being labeled with a reporter and terminating at its 5' end with a nucleotide containing a base selected from the group consisting of A, C, T, and G, and the 5' portion containing a quencher, such that the quencher interacts with the reporter to prevent the reporter from producing a signal; (v) a nucleic acid cleavage means; (b) mixing the target nucleic acid, the invading oligonucleotides, the primary cleavage oligonucleotides and the nucleic acid cleavage means in the array to create a reaction mixture under reaction conditions such that at least the 3' portion and middle portion of those primary cleavage oligonucleotides having a middle portion complementary to the SNP portion of the target nucleic acid are annealed to the target nucleic acid and wherein at least the 5' portion of the invading oligonucleotide is annealed to the target nucleic acid so as to create a cleavage structure, wherein cleavage of the cleavage structure occurs to generate cleaved assay specific probes comprising the 5' portion and middle portion of those primary cleavage oligonucleotides having a middle portion complementary to the SNP portion of the target nucleic acid, such that the cleaved assay specific probe terminates at its 3' end with a nucleotide complementary to the SNP portion of the target nucleic acid; (c) incubating the reaction mixture under reaction conditions such at least the 5' portion of the cleaved assay specific probe is annealed to the 3' portion of the capture oligonucleotide and the nucleotide at the 3' end of the cleaved assay specific probe is
annealed to the nucleotide at the 5' end of the middle portion of the capture oligonucleotide so as to create a secondary cleavage structure, wherein cleavage of the secondary cleavage structure occurs to thereby release the quencher from the capture oligonucleotide; (d) detecting signal from the reporter and associating it with its location on the array to identify the bar code sequence contained in the capture oligonucleotides from which the quencher was removed to thereby identify the SNP in the target nucleic acid. In another embodiment, the invention includes a method of detecting and identifying a single nucleotide polymorphism (SNP) and a gene locus in a target nucleic acid molecule in an assay, comprising: (a) providing: (i) a target nucleic acid molecule having a first portion, a second portion comprising a SNP, and a third portion, wherein the first portion is downstream of and contiguous to the second portion, the second portion is downstream of and contiguous to the third portion; (ii) an invading oligonucleotide, having a 3' portion and a 5' portion, the 5' portion of the invading oligonucleotide containing sequence complementary to the first portion of the target nucleic; (iii) at least one set of primary cleavage oligonucleotides for each gene locus, each primary cleavage oligonucleotide having a 3' portion complementary to the third portion of the target nucleic acid, a middle portion containing a nucleotide having a base selected from the group consisting of A, T, C, and G, and a 5' portion containing an SNP-specific bar code sequence and a gene locus-specific bar code sequence; (iv) an array comprising a set of capture oligonucleotides and a solid phase, each capture oligonucleotide having a 5' portion, a middle portion, and a 3' portion, the 3' portion being attached at the 3' end to the solid phase and containing sequences complementary the SNP-specific bar code sequence and the gene locus bar code sequence, wherein the bar code sequences of each capture oligonucleotide is associated with a location on the array, the middle portion having a sequence capable of forming a hairpin loop, the middle portion being labeled with a reporter and terminating at its 5' end with a nucleotide containing a base selected from the group consisting of A, C, T, and G, and the 51 portion containing a quencher, such that the quencher interacts with the reporter to prevent the reporter from producing a signal; (v) a nucleic acid cleavage means; (b) mixing the target nucleic acid, the invading oligonucleotides, the primary cleavage oligonucleotides and the nucleic acid cleavage means in the array to create a reaction mixture under reaction conditions such that at least the 3' portion and middle portion of those primary cleavage oligonucleotides having a middle portion complementary to the SNP portion of the target nucleic acid are annealed to the target nucleic acid and wherein at least the 5' portion of the invading oligonucleotide is annealed to the target nucleic acid so as to create a cleavage structure, wherein cleavage of the cleavage structure occurs to generate cleaved assay specific
probes comprising the 51 portion and middle portion of those primary cleavage oligonucleotides having a middle portion complementary to the SNP portion of the target nucleic acid, such that the cleaved assay specific probe terminates at its 3' end with a nucleotide complementary to the SNP portion of the target nucleic acid; (c) incubating the reaction mixture under reaction conditions such at least the 5' portion of the cleaved assay specific probe is annealed to the 3' portion of the capture oligonucleotide and the nucleotide at the 3' end of the cleaved assay specific probe is annealed to the nucleotide at the 5' end of the middle portion of the capture oligonucleotide so as to create a secondary cleavage structure, wherein cleavage of the secondary cleavage structure occurs to thereby release the quencher from the capture oligonucleotide; (d) detecting signal from the reporter and associating it with its location on the array to identify the bar code sequences contained in the capture oligonucleotides from which the quencher was removed to thereby identify the SNP and gene locus in the target nucleic acid. In another embodiment, the invention includes a method of detecting and identifying a single nucleotide polymorphism (SNP) at a specific locus in a target nucleic acid molecule in an assay, comprising: (a) providing: (i) a target nucleic acid molecule having a first portion, a second portion comprising a SNP, and a third portion, wherein the first portion is downstream of and contiguous to the second portion, the second portion is downstream of and contiguous to the third portion; (ii) an invading oligonucleotide having a 3' portion and a 5' portion, the 5' portion of the invading oligonucleotide containing sequence complementary to the first portion of the target nucleic; (iii) at least one set of primary cleavage oligonucleotides, each primary cleavage oligonucleotide having a 3' portion complementary to the third portion of the target nucleic acid, a middle portion containing a nucleotide having a base selected from the group consisting of A, T, C, and G, and a 5' portion containing a SNP-specific bar code sequence and a locus-specific bar code sequence; (iv) a nucleic acid cleavage means; (b) mixing the target nucleic acid, the invading oligonucleotides, the primary cleavage oligonucleotides and the nucleic acid cleavage means to create a reaction mixture under reaction conditions such at least the 3' portion and middle portion of those primary cleavage oligonucleotides having a middle portion complementary to the SNP portion of the target nucleic acid are annealed to the target nucleic acid and wherein at least the 5' portion of the invading oligonucleotide is annealed to the target nucleic acid so as to create a cleavage structure, wherein cleavage of the cleavage structure occurs to generate cleavage products comprising the 5' portion and middle portion of those primary cleavage oligonucleotides having a middle portion complementary to the SNP portion of the target nucleic acid; and (c) detecting the SNP- specific sequence and the locus-specific sequence in the cleavage products to thereby identify the SNP and the locus in the target nucleic acid.
In another embodiment, the invention includes a method of detecting and identifying a single nucleotide polymorphism (SNP) at a specific locus in a target nucleic acid molecule in an assay, comprising: (a) providing: (i) a target nucleic acid molecule having a first portion, a second portion comprising a SNP, and a third portion, wherein the first portion is downstream of the second portion, the second portion is downstream of and contiguous to the third portion; (ii) an invading oligonucleotide having a 3' portion and a 5' portion, the 5' portion of the invading oligonucleotide containing sequence complementary to the first portion of the target nucleic; (iii) a set of primary cleavage oligonucleotides comprising four unique oligonucleotides for each locus of interest, such that each primary cleavage oligonucleotide contains a unique sequence ("bar code") denoting a specific locus and a specific SNP, each primary cleavage oligonucleotide having a 3' portion complementary to the third portion of the target nucleic acid, a middle portion containing a nucleotide selected from the group consisting of A, T, C, and G, and a 5' portion containing the bar code; (iv) a nucleic acid cleavage means; (b) mixing the target nucleic acid, the invading oligonucleotides, the primary cleavage oligonucleotides and the nucleic acid cleavage means to create a reaction mixture under reaction conditions such at least the 3' portion and middle portion of those primary cleavage oligonucleotides having a middle portion complementary to the SNP portion of the target nucleic acid are annealed to the target nucleic acid and wherein at least the 5' portion of the invading oligonucleotide is annealed to the target nucleic acid so as to create a cleavage structure, wherein cleavage of the cleavage structure occurs to generate cleaved assay specific probes comprising the 5' portion and middle portion of those primary cleavage oligonucleotides having a middle portion complementary to the SNP portion of the target nucleic acid; (c) providing an array comprising a set of capture oligonucleotides and a solid phase, each capture oligonucleotide having a 5' portion, a middle portion, and a 3' portion, the 3' portion being attached at the 3' end to the solid phase and containing sequence complementary to the bar code, the middle portion having a sequence capable of forming a hairpin loop, the middle portion being labeled with a reporter and terminating at its 5' end with a nucleotide selected from the group consisting of A, C, T, and G, and the 5' portion contain a quencher, such that the quencher interacts with the reporter to prevent the reporter from producing a signal; (d) adding the cleaved assay specific probe from the reaction mixture to the array to create an array mixture under reaction conditions such at least the 5' portion of the cleaved assay specific probe is annealed to the 3' portion of the capture oligonucleotide and the nucleotide at the 3' end of the cleavage product is annealed to the nucleotide at the 5' end of the middle portion of the capture oligonucleotide so as to create a secondary cleavage structure, wherein cleavage of the secondary cleavage structure occurs to release the quencher from the capture
oligonucleotide; (e) detecting signal from the reporter to identify those capture oligonucleotides from which the quencher was removed to thereby identify the SNP and locus in the target nucleic acid. In another embodiment, the invention includes a method of detecting and identifying a single nucleotide polymorphism (SNP) at a specific locus in a target nucleic acid molecule in an assay, comprising: (a) providing: (i) a target nucleic acid molecule having a first portion, a second portion comprising an SNP, and a third portion, wherein the first portion is downstream of the second portion, the second portion is downstream of and contiguous to the third portion; (ii) an invading oligonucleotide having a 3' portion and a 5' portion, the 5' portion of the invading oligonucleotide containing sequence complementary to the first portion of the target nucleic; (iii) a set of primary cleavage oligonucleotides comprising four unique oligonucleotides for each locus of interest, such that each primary cleavage oligonucleotide contains a unique sequence ("bar code") denoting a specific locus and a specific SNP, each primary cleavage oligonucleotide having a 3' portion complementary to the third portion of the target nucleic acid, a middle portion comprising a nucleotide selected from the group consisting of A, T, C, and G, and a 5' portion containing the bar code and a detectable label; (iv) a nucleic acid cleavage means; (b) mixing the target nucleic acid, the invading oligonucleotides, the primary cleavage oligonucleotides and the nucleic acid cleavage means to create a reaction mixture under reaction conditions such at least the 3' portion and middle portion of those primary cleavage oligonucleotides having a middle portion complementary to the SNP portion of the target nucleic acid are annealed to the target nucleic acid and wherein at least the 5' portion of the invading oligonucleotide is annealed to the target nucleic acid so as to create a cleavage structure, wherein cleavage of the cleavage structure occurs to generate cleaved assay specific probes comprising the 5' portion and middle portion of those primary cleavage oligonucleotides having a middle portion complementary to the SNP portion of the target nucleic acid; and (c) detecting the bar code in the cleaved assay specific probe to thereby identify the SNP and the locus in the target nucleic acid. In another embodiment, the invention includes a method of detecting and identifying a single nucleotide polymorphism (SNP) at a specific locus in a target nucleic acid molecule in an assay, comprising: (a) providing: (i) a target nucleic acid molecule having a first portion, a second portion comprising a SNP, and a third portion, wherein the first portion is downstream of the second portion, the second portion is downstream of and contiguous to the third portion; (ii) an invading oligonucleotide having a 3' portion and a 5' portion, the 5' portion of the invading oligonucleotide containing sequence complementary to the first portion of the target nucleic; (iii) a set of primary cleavage oligonucleotides comprising four unique oligonucleotides for each locus of interest, such
that each primary cleavage oligonucleotide contains a unique sequence ("bar code") denoting a specific locus and a specific SNP, each primary cleavage oligonucleotide having a 3' portion complementary to the third portion of the target nucleic acid, a middle portion comprising a nucleotide selected from the group consisting of A, T, C, and G, and a 5' portion containing the bar code and a detectable label; (iv) a nucleic acid cleavage means; (b) mixing the target nucleic acid, the invading oligonucleotides, the primary cleavage oligonucleotides and the nucleic acid cleavage means to create a reaction mixture under reaction conditions such at least the 3' portion and middle portion of those primary cleavage oligonucleotides having a middle portion complementary to the SNP portion of the target nucleic acid are annealed to the target nucleic acid and wherein at least the 5' portion of the invading oligonucleotide is annealed to the target nucleic acid so as to create a cleavage structure, wherein cleavage of the cleavage structure occurs to generate cleaved assay specific probes comprising the 5' portion and middle portion of those primary cleavage oligonucleotides having a middle portion complementary to the SNP portion of the target nucleic acid; (c) providing an array comprising a set of capture oligonucleotides and a solid phase, each capture oligonucleotide being attached at one end to the solid phase and containing sequence complementary to the bar code, wherein the location of each capture oligonucleotide on the solid phase is correlated with the bar code contained in the capture oligonucleotide; (d) adding the cleaved assay specific probe from the reaction mixture to the array to create an array mixture under reaction conditions such that at least some portion of the cleaved assay specific probe is annealed to those capture oligonucleotides containing sequence complementary to the bar code sequence contained in the cleaved assay specific probe; (e) removing unannealed cleaved assay specific probe; and (f) detecting the label on annealed cleaved assay specific probe, correlating the position of the annealed cleaved assay specific probe on the array to the bar code of the capture oligonucleotide, to thereby identify the SNP and locus in the target nucleic acid. In another embodiment, the invention includes a method of monitoring the specific activity of a nucleic acid cleavage means, comprising: (a) providing: (i) a target nucleic acid molecule having a first portion, a second portion, and a third portion, wherein the first portion is downstream of the second portion, the second portion is downstream of and contiguous to the third portion; (ii) an invading oligonucleotide having a 3' portion and a 5' portion, the 5' portion of the invading oligonucleotide containing sequence complementary to the first portion of the target nucleic; (iii) a primary cleavage oligonucleotide, each primary cleavage oligonucleotide having a 5' portion containing a first detectable label and a 3' portion complementary to the third portion of the target nucleic acid and containing a second detectable label; (iv) a nucleic acid cleavage means; (b) mixing the target nucleic acid, the invading oligonucleotide, the primary cleavage
oligonucleotide and the nucleic acid cleavage means to create a reaction mixture under reaction conditions such that at least the 3' portion of the primary cleavage oligonucleotide is annealed to the target nucleic acid and wherein at least the 5' portion of the invading oligonucleotide is annealed to the target nucleic acid so as to create a cleavage structure, wherein cleavage of the cleavage structure occurs to generate a cleavage product comprising the 5' portion of the primary cleavage oligonucleotide and the first label; (c) denaturing the reaction mixture; (d) providing an array comprising a capture oligonucleotide and a solid phase, the capture oligonucleotide being attached at one end to the solid phase and containing sequence complementary to the 5' portion of the primary cleavage oligonucleotide; (e) adding the cleavage products from the reaction mixture to the array under reaction conditions such that the cleavage product containing the 5'end and first label as well as uncleaved primary cleavage oligonucleotide containing both the first and the second label is annealed to the capture oligonucleotide, and washing the array to remove unannealed cleavage product; (f) measuring the signal intensity of the first label to thereby provide a measure of the total amount of cleavage product containing the 5'end and first label as well as the uncleaved primary cleavage oligonucleotide containing both the first and the second label; (g) measuring the signal intensity of the second label to thereby provide a measure of the uncleaved primary cleavage oligonucleotide containing both the first and the second label; (h) calculating the amount of cleavage product containing the 5'end and first label and determining the ratio of cleavage product containing the 5'end and first label to cleavage product containing the 5'end and first label plus uncleaved primary cleavage oligonucleotide to thereby provide a measure of the specific acitivity of the nucleic acid cleavage means. In another embodiment, the invention includes a kit for detecting and identifying a single nucleotide polymorphism (SNP) at a specific locus in a target nucleic acid molecule in an assay, the kit comprising: a set of primary cleavage oligonucleotides corresponding to the target nucleic acid comprising four unique oligonucleotides for each locus of interest, such that each primary cleavage oligonucleotide contains a unique sequence ("bar code") denoting a specific locus and a specific SNP, each primary cleavage oligonucleotide having a 3' portion complementary to the target nucleic acid, a middle portion comprising a nucleotide selected from the group consisting of A, T, C, and G, and a 5' portion containing the bar code and a detectable label; an invading oligonucleotide corresponding to the target nucleic acid; a nucleic acid cleavage means for cleaving a primary cleavage structure formed by the primary cleavage oligonucleotide, the invading oligonucleotide and the target nucleic acid to form a cleaved assay specific probe; an array comprising a set of capture oligonucleotides and a solid phase, each capture oligonucleotide being attached at one end to the solid phase and containing sequence
complementary to the cleaved assay specific probe, including the bar code, wherein the location of each capture oligonucleotide on the solid phase is correlated with the bar code contained in the capture oligonucleotide. These and other objects and features of the invention will be more fully appreciated when the following detailed description of the invention is read in conjunction with the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram of one embodiment of the system and method for detecting a target nucleic acid molecule of the present invention. FIG. 2 is an illustration of a 4x4 array of capture oligonucleotides deposited in a well of an activated polypropylene plate. FIG. 3 is a flow chart illustrating a method for analyzing fluorescence changes in capture oligonucleotides deposited onto a solid substrate according to an embodiment of the present invention. FIG. 4 is a schematic diagram of an embodiment of the present invention wherein an assay specific probe is cleaved from a primary cleavage structure and hybridized to a capture oligonucleotide coupled to a fluorescent label and deposited onto a solid substrate. FIG. 5 is an illustration of two primary cleavage oligonucleotides, each having both a gene loci-specific bar code sequence and a bar code sequence specific for the nucleotide A at the site of an SNP. FIG. 6 is an illustration of two primary cleavage oligonucleotides, each having both an SNP-specific bar code sequence and a bar code sequence specific for gene loci 1. FIG. 7 is a schematic diagram of an embodiment of the present invention wherein a 5'-labeled assay specific probe is cleaved from a primary cleavage structure and hybridized to a capture oligonucleotide deposited onto a solid substrate. FIG. 8 is a schematic diagram illustrating Enzyme-Labeled Fluorescence (ELF) signal amplification according to an embodiment of the present invention using labeled primary cleavage oligonucleotides. FIG. 9 is a schematic diagram illustrating the use of a dual-labeled primary cleavage oligonucleotide according to the practice of one embodiment of the present invention. FIG. 10 is a schematic diagram illustrating the use of paramagnetic or magnetic beads and a magnet to scavenge uncleaved affinity-tagged primary cleavage oligonucleotides following a cleavage reaction. FIG. 11 is a schematic diagram and photograph showing the effect of primary cleavage oligonucleotide concentration on the effectiveness of the removal of uncleaved
reactants. FIG. 12 is a schematic diagram and table showing the effectiveness of using biotin-tagged dual labeled primary cleavage oligonucleotide with magnetic bead capture to remove uncleaved reaction product.
DETAILED DESCRIPTION OF THE INVENTION Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs. Although any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods, devices and materials are now described. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing, for example, the oligonucleotides, constructs, and methodologies that are described in the publications which might be used in connection with the presently described invention. The publications discussed above and throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention.
Definitions The terms "polynucleotide" or "nucleic acid" and their respective plurals are used essentially interchangeably herein and are intended to include naturally occurring or synthesized double stranded deoxyribonucleic acid (hereinafter "DNA"), single stranded DNA, or ribonucleic acid (hereinafter "RNA"). The terms "complementary" or "complementarity" as used herein refer to polynucleotides that undergo Watson-Crick base pairing. This includes the pairing of "nucleoside analogs", such as deoxyinosine, nucleosides with 2-aminopurine bases, and the like, that may be employed. As used herein, "nucleoside" includes the natural nucleosides, including 2'-deoxy and 2'-hydroxyl forms, e.g. as described in Kornberg and Baker, DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992). "Analogs" in reference to nucleosides includes synthetic nucleosides having modified base moieties and/or modified sugar moieties that are capable of specific hybridization, e.g. described by Scheit, Nucleotide Analogs (John Wiley, New York, 1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990), or the like. Such analogs include synthetic nucleosides designed to enhance binding properties, reduce degeneracy, increase specificity, and the like. Total or perfect complementarity as used herein means that the polynucleotide or
oligonucleotide strands making up a duplex form a double stranded structure with one another such that every nucleotide in each strand undergoes Watson-Crick base pairing with a nucleotide in the other strand and that there are no mismatches. "Partially complementary" as used herein includes polynucleotide or oligonucleotide strands containing unmatched and non-hybridizing nucleotides, but in which the remaining "matched" nucleotides undergo Watson-Crick base pairing. A "self-complementary" nucleic acid is one in which separate regions of the nucleic acid are capable of base-pairing with one another to forøi, for example, a hairpin structure. "Homology" refers to a degree of identity between two or more nucleic acid sequences. There may be partial homology or complete homology. A partially identical sequence is one that is less than 100% identical to another sequence. "Hybridization" is used herein in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization is affected by variables such as the degree of complementary between the nucleic acids, stringency of the hybridization conditions involved, the T (melting temperature) of the formed hybrid, and the G:C ratio within the nucleic acids. The term "oligonucleotide" as used herein is defined as a molecule comprised of two or more 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. Such oligonucleotides may be generated by various methods, including chemical synthesis, DNA replication, reverse transcription, or a combination thereof. 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. As referred to herein, 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 "downstream" region when moving along a strand of nucleic acid in a 5' to 3' direction. "Target nucleic acid" as used herein may comprise single or double-stranded DNA or RNA. Target nucleic acids contain a sequence that has at least partial complementarity
with at least a primary cleavage oligonucleotide, and may also have at least partial complementarity with an invading oligonucleotide. "Primary cleavage oligonucleotide" refers to an oligonucleotide which contains a flap portion and a non-flap portion, the non-flap portion having a least partial complementarity with the target nucleic acid, and the flap portion optionally having one or more bar code sequences. "Bar code sequence" refers to a specific sequence of nucleotides selected so as to identify a specific feature of the target nucleic acid. Bar code sequences typically are nonsense or unrelated sequence to that of the gene of interest in the target nucleic acid, designed for specific (perfect match) hybridization to a region of the solid phase tethered capture oligonucleotide. Examples include a locus bar code sequence, in which different gene loci are assigned different bar code sequences, and a single nucleotide polymorphism ("SNP") bar code, in which different SNPs are given different bar code sequences. Other examples of useful bar code sequences will be apparent to one of skill in the art. "Invading oligonucleotide" refers to an oligonucleotide which contains sequences at its 3' end which are substantially the same as sequences located at the 5' end of the non- flap portion of a primary cleavage oligonucleotide; these regions will compete for hybridization to the same segment along a complementary target nucleic acid. A "capture oligonucleotide" as used herein refers to an oligonucleotide that is complementary and capable of hybridizing to at least a portion of another oligonucleotide or nucleic acid. Capture oligonucleotides can be immobilized to a solid support such that an oligonucleotide or nucleic acid that binds to the capture oligonucleotide becomes bound or tethered to the solid support. An oligonucleotide is said to be present in "excess" relative to another nucleic acid molecule if that oligonucleotide is present at a higher molar concentration than the other nucleic acid molecule. As an example, when a primary cleavage oligonucleotide is present in excess of a target nucleic acid it will be present in at least a 10 to 100-fold molar excess; typically at least 1 pmole of each primary cleavage oligonucleotide would be used when the target nucleic acid sequence is present at about 10 frnoles or less. See, for example, U.S. Patent No. 6,090,543. The term "nucleic acid sequence" as used herein refers to an oligonucleotide, nucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin which may be single or double-stranded, and represent the sense or antisense strand. Overview. An overview of a system and method according to a preferred embodiment is shown in FIG. 1, and will now be described to illustrate the various components of the
system. A target nucleic acid 10, is hybridized to a primary cleavage oligonucleotide 12. An invading oligonucleotide 14 also hybridizes to the target nucleic acid 10 to form a primary cleavage structure. A structure-specific nuclease cleaves the primary cleavage oligonucleotide 12, releasing a cleaved assay specific probe 16. The cleaved assay specific probe 16 hybridizes to a capture oligonucleotide 18 having a fluor 20 and a quencher 22. The capture oligonucleotide is coupled to a solid phase 24 in an array such that a specific location 26 in the array is associated with a specific target nucleic acid. Upon hybridization of the cleaved assay specific probe 16 to the capture oligonucleotide 18, a secondary cleavage structure is formed, and upon addition of nucleic acid cleavage means, the quencher 20 is released allowing the fluor to be capable of fluorescent resonance energy transfer (FRET). The solid substrate is then illuminated, generating emitted fluorescent light at any location where the quencher has been released. The resulting fluorescence is detected and the position in the array is mapped and conelated to known target nucleic acid positions to thereby identify the specific target nucleic acid present in the sample. Target Nucleic Acids The invention provides means for forming nucleic acid cleavage structures that are dependent upon the presence of a target nucleic acid. The nucleic acid cleavage structures are cleaved by a nucleic acid cleavage means to release distinct cleaved assay specific probes corresponding to each target nucleic acid. In a typical embodiment, one or more target nucleic acids are derived from one or more samples. Each sample is known to contain, or is suspected of containing, one or more target nucleic acids. Target nucleic acids include various types of both RNA and DNA (including single stranded DNA and double stranded DNA) that are obtained according to standard techniques known in the art. For example, samples containing nucleic acids may be obtained from a tissue sample, tissue culture cells, samples containing bacteria and/or viruses, etc. Also, the target nucleic acid may also be transcribed in vitro from a DNA template or may be synthetic, chemically synthesized, or generated via polymerase chain reaction (PCR). Further, nucleic acids may be isolated from organisms in the form of genomic material, plasmids, or similar extrachromosomal DNA, or the nucleic acid may be a fragment of such material generated by treatment with a restriction endonuclease or other cleavage agent. The binding of invading oligonucleotides 14 and primary cleavage oligonucleotides 12, described in more detail below, divides the target nucleic acid 10 into three distinct regions, as shown in FIG. 1. A first region 28, has complementarity to only the invading oligonucleotide 14; a second region 30, has complementarity to both the invading oligonucleotide 14 and the primary cleavage oligonucleotide 12; and a third
region 32, has complementarity only to a portion of the corresponding primary cleavage oligonucleotide 12. The first region of each target nucleic acid 10 is contiguous to and downstream from the second region, and the second region of each target nucleic acid is contiguous to and downstream from the third region. A target nucleic acid may be either single-stranded or double-stranded. Double- stranded target nucleic acids can be rendered single stranded, for example by heating. In the methods described herein, the target nucleic acid can be reused or recycled during multiple rounds of hybridization with oligonucleotides and cleavage means. In a preferred embodiment, the methods of the invention are used to detect single nucleotide polymorphisms (SNP's). hi the examples described herein, the methods of the present invention used the Factor V (Leiden) SNP. Other targets are known in the art and include, but are not limited to, Apolipoprotein E (ApoE)(C112R); Apolipoprotein E (ApoE)(R158C); Factor II (G320210A); Factor V (Leiden)(G1691A); Glycoprotein la (Gpla)(C807T); Glycoprotein Ilia (PL A1/A2)(T565C); Mehtylenetetrahydrofolate Reductase (MTHFR) (A1298C); Methlenetetrahydrofolate Reductase (MTHFR) (C677T) Plasminogen Activator Inhibitor-1 (PAL-1); ATP -Binding Cassette, Subfamily B Member 1; Cytochrome P450, Subfamily 1A, Polypeptide 1; Cytochrome P450 Subfamily 1A, Polypeptide 2; Cytochrome P450, Subfamily IIB, Polypeptide 6 Cytochrome P450, Subfamily I IC, Polypeptide 9, Glucuronidase, Beta; Interferon Gamma; Interleukin 1, Beta; ϊnterleukin 2; Interleukin 4; Interleukin 6; Interleukin 8 Interleukin 10; Involucrin; Matrix Metalloproteinase 3; Nuclear Factor of Kappa Light Polypeptide Gene Enhancer in B-cells Inhibitor, Alpha; Nuclear Receptor Subfamily 3, Group C, Member 1; Oncostatin M; Prostaglandin-Endoperoxide Synthase 2; Small Inducible Cytokine A2; Solute Carrier Family 21(organic anion transporter), Member 9 (hSLC21A9); Sulfotransferase Family, Cytosolic, 1A, Phenol-Preferring, Members 1-3, Pan Detection; Transforming Growth Factor, Beta 1; Tumor Necrosis Factor (TNF Superfamily, Member 2); v-fos FBJ murine osteosarcoma viral oncogene homolog; and v- myc myelocytomatosis viral oncogene homolog (avian). Known SNPs are reported in the literature. See, for example, Mein, et al. (2000) Genome Research 10: 330-343, hereby incorporated by reference in its entirety. Primary Cleavage Structure The methods described herein typically utilize at least a pair of oligonucleotides that interact with each target nucleic acid to form a primary cleavage structure that is a substrate for a structure-specific nuclease. For each target nucleic acid 10, there is a corresponding primary cleavage oligonucleotide 12. As shown in FIG. 1, each primary cleavage oligonucleotide comprises a target-specific (or non-flap) portion 34 and a cleavage (or flap) portion 36,
located upstream of the target specific portion. The target-specific portion 34 of each primary cleavage oligonucleotide defines the third region 32 of the target nucleic acid sequence by being the complement of that region for a corresponding target nucleic acid. The cleavage portion 36 of each primary cleavage oligonucleotide defines the second region 30 of the target nucleic acid sequence by being the complement of that region for a corresponding target nucleic acid. The target nucleic acid strand that anneals with the primary cleavage oligonucleotide does so in a typical anti-parallel orientation. For each target nucleic acid, there is a conesponding invading oligonucleotide 14. Each invading oligonucleotide comprises two portions, a cleavage portion and a target- specific portion. The cleavage portion of an invading oligonucleotide comprises a sequence complementary to the second region 30 of the corresponding target nucleic acid 10. Thus, for each particular target nucleic acid 10, the cleavage portion of an invading oligonucleotide 14 invades the cleavage portion 36 of the primary cleavage oligonucleotide. The invading oligonucleotide 14 is located upstream of the primary cleavage oligonucleotide 12 with respect to the target nucleic acid strand 10. Both the invading oligonucleotide 14 and the primary cleavage oligonucleotide 12 anneal to the corresponding target nucleic acid 10 in an anti-parallel orientation to the target nucleic acid strand. The target-specific portion of the invading oligonucleotide 14 comprises a sequence complementary to the first region 28 of the conesponding target nucleic acid 10. The invading oligonucleotide 14 and the primary cleavage oligonucleotide 12 are arranged in a parallel orientation relative to one another. The oligonucleotides of the invention are designed in accordance with methods that are known in the art. An initial consideration in choosing the length of oligonucleotides is the temperature under which they will be expected to be utilized for the methods described herein. For example, the chosen length of an oligonucleotide might vary depending on the thermal stability of the cleavage means. Longer oligonucleotides are generally expected to have a higher hybridization specificity. It is desirable that the oligonucleotides of the invention have a length that is long enough to be reasonably expected to hybridize only to the intended target sequence within a complex sample. The oligonucleotide probes of the invention are typically 10 to 40 nucleotides in length, and more typically 25 to 35 nucleotides in length. See Lyamichev et al. (1999) Nature Biotechnology 17: 292-296; see also, U.S. Patent Nos. 6,562,611,5,994,069 and 5,846,717, the entire contents of which are hereby incorporated by reference. Where only a portion of a particular oligonucleotide is expected to anneal to a target nucleic acid strand, the length of the annealing portions are typically 10 to 40 nucleotides in length, and more typically 15 to 25 nucleotides in length. It is not intended that the method of the present invention be limited to any particular size of the primary
cleavage oligonucleotide or invading oligonucleotide. An important feature of the method is that the primary cleavage oligonucleotide can depart from the target nucleic acid after being cleaved, thus permitting the annealing and cleavage of other copies of the primary cleavage oligonucleotides ("turnover") without any discrete denaruration or displacement steps. One characteristic which is important in determining turnover is the melting temperature (Tm) of the primary cleavage oligonucleotide. This is effected both by length and G+C content of the oligonucleotide. In general, oligonucleotides with longer lengths and/or higher G+C content have higher melting temperatures. Following cleavage, the cleavage (flap) portion of the primary cleavage oligonucleotide is released, leaving the target-specific (non-flap) portion annealed to the target nucleic acid. If the Tm of target-specific portion of the primary cleavage oligonucleotide is less than the reaction temperature, and the reaction temperature is less than the Tm of the entire primary cleavage oligonucleotide, then cleavage of the primary cleavage oligonucleotide will lead to the departure of target-specific portion of the primary cleavage oligonucleotide and a new primary cleavage oligonucleotide will be able to hybridize. In one embodiment, the primary cleavage oligonucleotide is designed so that after cleavage of a portion of the primary cleavage oligonucleotide, the Tm of the remainder of the primary cleavage oligonucleotide is below the reaction temperature. Another factor affecting hybridization of the primary cleavage oligonucleotide is competition from the invading oligonucleotide. Preferably, the portions of the primary cleavage and invading oligonucleotides that anneal with the first and third regions of the nucleic acid target are selected so as to have similar melting temperatures. If the binding of the cleavage portion of the invading oligonucleotide to the target nucleic acid is more stable than the binding of the cleavage portion of the primary cleavage oligonucleotide, then the cleavage portion of the invading oligonucleotide may be favored in the competition for binding to the second region of the target. Alternatively, if the target- specific portion of primary cleavage oligonucleotide binds particularly strongly to the target nucleic acid, then the invading oligonucleotide will still cause internal cleavage of the primary cleavage oligonucleotide, but the target-specific portion of the primary cleavage oligonucleotide bound to the third region of the target nucleic acid may not dissociate at the reaction temperature. This is likely to reduce the detection signal because the turnover would be less than optimal. Another objective in optimizing the cleavage assay is to allow specific detection of the fewest copies of a target nucleic acid. To achieve this end, it is desirable that the combined elements of the reaction interact with the maximum efficiency, so that the rate of the reaction (e.g., the number of cleavage events per minute) is maximized. Elements
contributing to the overall efficiency of the reaction include the rate of hybridization, the rate of cleavage, and the efficiency of the release of the cleaved probe. The rate of cleavage will be a function of the cleavage means chosen, and may be made optimal according to the manufacturer's instructions when using commercial preparations of enzymes or as described in the examples herein. The other elements (rate of hybridization, efficiency of release) depend upon the execution of the reaction. Three elements of the cleavage reaction that significantly affect the rate of nucleic acid hybridization are the concentration of the nucleic acids, the temperature at which the cleavage reaction is performed and the concentration of salts and/or other charge- shielding ions in the reaction solution. Methods for optimizing these conditions are well known in the art. Generally, the one or more samples comprising target nucleic acid, and in preferred embodiments more than one target nucleic acid, primary cleavage oligonucleotide(s), and invading oligonucleotide(s) are assembled into a buffered reaction mixture suitable for nucleic acid modification enzymes, the components of which are well known to those of skill in the art. Typically, the primary cleavage oligonucleotides are provided in sufficient excess (generally, at least a 100-fold molar excess) so that the rate of hybridization to the target nucleic acid(s) is rapid. The reactions are typically performed with at least 1 pmole of each primary cleavage oligonucleotide when the target nucleic acid sequence is present at about 10 fmoles or less. Other oligonucleotide concentrations, either higher or lower, commonly used in the art are contemplated and the methods described herein are not limited to these amounts. It is advantageous for invading oligonucleotides to be immediately available to direct the cleavage of each primary cleavage oligonucleotide that hybridizes to a target nucleic acid. However, because the invading oligonucleotide is not cleaved and is reusable, the primary cleavage oligonucleotide is provided in excess of the invading oligonucleotide in the reaction. An exemplary ratio of primary cleavage oligonucleotide to invading oligonucleotide are between a 2 and a 1000 fold excess of primary cleavage oligonucleotide over invading oligonucleotide. These ratios are not intended to limit the scope of the invention and other ratios may be employed. Nucleic Acid Cleavage Means A nucleic acid cleavage means encompasses any means that is capable of cleaving a cleavage structure, including but not limited to enzymes. Typically, the nucleic acid cleavage means comprise one or more "structure-specific nucleases". These structure- specific nucleases recognize specific secondary structures in a nucleic acid molecule and cleave these structures. The nucleic acid cleavage means includes native DNA polymerases having 5'
nuclease activity (e.g., Taq DNA polymerase, E. coli DNA polymerase I), nuclease activity provided from a variety of sources including the Cleavase enzymes, the FEN-1 endonucleases (including RAD2 and XPG proteins; AfuFEN, PfuFEN, MjaFEN, MthFEN), Taq and Tth DNA polymerases (including TaqPol, TaqExo, and TthPol) and E. coli DNA polymerase I. In preferred embodiments the cleavage means includes DNA polymerases that have been modified so as to have a 5' nuclease without synthetic activity. The nucleic acid cleavage means further encompasses 5' nucleases derived from thermostable Type A DNA polymerases that retain 5' nuclease activity but have reduced or absent synthetic activity. Modified enzymes having reduced synthetic activity and increased 5' nuclease activity relative to unmodified enzymes are also envisioned by the present invention. Typically, the modified enzyme may have no synthetic activity remaining or may have that level of synthetic activity that will not interfere with the use of the modified enzyme in the detection assay described below. Exemplary nucleic acid cleavage means are known in the art, and are described in more detail in Kaiser et al. (1999) J. Biol. Chem. 274: 21387-21394; U.S. Patent No. 5,994,069 to Hall et al., U.S. Patent No. 5, 719, 028 to Dahlberg et al., U.S. Patent No. 5,837,450 to Dahlberg et al., the contents of which are all hereby incorporated by reference in their entirety. Primary Stage Cleavage A "cleavage structure" refers to a structure that is formed by the interaction of one or more oligonucleotides and a target nucleic acid to form a duplex, where the resulting structure is cleavable by a cleavage means. The cleavage structure is a substrate for specific cleavage by the cleavage means, as opposed to a nucleic acid molecule that is a substrate for non-specific cleavage by agents such as phosphodiesterases that cleave nucleic acid molecules without regard to secondary structure (i.e., no formation of a duplexed structure is required). The products generated by the reaction of a cleavage means with a cleavage structure are referred to herein as "cleavage products". A primary cleavage structure is generated for each target nucleic acid by the interaction of each target nucleic acid with the corresponding primary cleavage oligonucleotide and the corresponding invading oligonucleotide. In each primary cleavage structure, at least the 3' portion of each primary cleavage oligonucleotide is annealed to a corresponding target nucleic acid. As described above, the 3' portion of the primary cleavage oligonucleotide anneals to the third region of the corresponding target nucleic acid. At least the 5' portion of each invading oligonucleotide is annealed to a first region of the corresponding target nucleic acid. The primary cleavage structures are cleaved by the nucleic acid cleavage means to
generate a cleavage product termed herein as a "cleaved assay specific probe". There is a unique cleaved assay specific probe formed conesponding to each target nucleic acid. Each cleaved assay specific probe comprises a 5' region and a 3' region. Secondary Cleavage In one embodiment of the instant invention, a secondary cleavage reaction is performed where the cleavage product of the first stage interacts with a unique capture oligonucleotide for each target and forms a secondary cleavage structure. Subsequent sequential cleavage reactions are within the scope of the invention, including tertiary cleavage reactions and so forth. The cleavage of the secondary cleavage structure or formation of second stage cleavage products can be used as a basis of detection or to drive further stages of sequential cleavage reactions. Importantly, the product of the secondary cleavage reaction is not capable of initiating either of the first or second stage cleavage reactions. In one reaction format, the reaction components for the first stage cleavage are mixed with components for the second stage cleavage reaction so that second stage reactions can be initiated directly after product from the primary cleavage reaction becomes available. In this format the primary and secondary cleavage events can take place simultaneously. The reaction format can also be configured with each step of cleavage reactions being spatially or temporally separated, such as performing each stage in different reaction vessels or by causing a change in reaction conditions that allow later cleavage events to take place. As shown in FIG. 1, in a prefened embodiment, each capture oligonucleotide 18 comprises contiguous first and second regions. The first region 38 comprises the 3' end of the capture oligonucleotide. The second region 40 comprises the 5' end of the capture oligonucleotide. The second region of the capture oligonucleotide further comprises a region of self-complementarity that is capable of forming a hairpin loop. For each specific target nucleic acid, the first region of each capture oligonucleotide is complementary to a corresponding cleaved assay specific probe that is generated in the primary cleavage reaction. Typically, a portion of the second region of each capture oligonucleotide is complementary to at least a portion of the 3' region of the corresponding cleaved assay specific probe. Upon contacting the products of the first cleavage reaction to the immobilized capture oligonucleotides, a secondary cleavage structure is formed for those capture oligonucleotides having cleaved assay specific probe hybridized thereto. Solid Substrate and Array In one aspect of the present invention, capture oligonucleotides are deposited onto a solid substrate, with the location of each type of capture oligonucleotide mapped to a
known position on the surface of the substrate. By providing capture oligonucleotides specific for each target nucleic acid, the particular target nucleic acid present in a sample can be determined by reference to the location of the fluorescent or other detectable signal generated on the surface of the substrate. As will be appreciated by one of skill in the art, this is an improvement over prior assays in which separate labels were required for each target nucleic acid in order to identify targets within a multiplexed sample. One type of solid substrate useful in the practice of the present invention is shown in FIG. 2. In this embodiment, capture oligonucleotides are deposited in a 4 x 4 array 42 in each well 44 of an activated microplate, such as an activated polypropylene plate 46. On each activated plate, wells are arranged in rows 48 and columns 50. In FIG. 2, there are 8 rows labeled A-H and 12 columns labeled 1-12; with a 4 x 4 array deposited in each well, such a plate could contain a total of 8 x 12 x 16 or 1,536 different types of capture oligonucleotides, each specific for a particular target nucleic acid. As will be appreciated by the skilled artisan, such an arrangement facilitates high through-put screening of multiple samples and automated detection and analysis. As will further be appreciated by the skilled artisan, such an array is one of many types of arrangements contemplated by this invention, which is not limited to the 4 x 4 array shown in FIG. 2, but which covers any anangement of capture oligonucleotides suitable for individual detection as described herein. 5'-arnino terminated capture oligonucleotides can be immobilized in the bottom of the wells of an acyl fluoride (ACF) activated polypropylene microplate using acyl fluoride surface activation, as described below in Example 3. Other means for attaching amino- oligonucleotide capture probes are known in the art, and may be used with the present invention. These include, but are not limited to, the use of epoxide, aldehyde or imidazole chemistries. Other examples are shown in U.S. Patent No. 6,146,833, the content of which is hereby incorporated by reference in its entirety.. The amount of capture oligonucleotide deposited at each predetermined site on the solid substrate is typically in the femtomole to picomoles range per reaction. Other oligonucleotide concentrations, either higher or lower, commonly used in the art are contemplated and the methods described herein are not limited to these amounts. Detection In one embodiment of the present invention, each capture oligonucleotide typically comprises a detectable label or moiety, generally placed at or near the 5' end of the secondary cleavage oligonucleotide, or may be positioned anywhere along the oligonucleotide. The detectable label can be dye such as a fluorophore. In preferred embodiments, the capture oligonucleotides comprise a fluorescent donor and fluorescent acceptor that generate a fluorescence-based signal in response to a change in distance
between the fluorescent donor and acceptor that is caused by cleavage. In preferred embodiments, the 5' end of each capture oligonucleotide comprises one or more detectable labels. In the preferred embodiment illustrated in Figure 1, the 5' end of each capture oligonucleotide comprises a fluorescent donor and fluorescent acceptor that generate a fluorescence-based signal in response to cleavage of the secondary cleavage structure. Various methods of detecting nucleic acids by using combinations of a fluorophore and an interacting molecule or moiety are described in Morrison, L. E., Haider, T. C. and Stols, L. M., A Solution phase detection of polynucleotides using interacting fluorescent labels and competitive hybridization, Analyt. Biochem. 183, 231- 244 (1989); Morrison, L. E. and Stols, L. M., A Sensitive fluorescence-based thermodynamic and kinetic measurements of DNA hybridization in solution, Biochemistry 32, 3095-3104 (1993); Gelfand et al. U.S. Pat. No. 5,210,015, and Livak et al. U.S. Pat. No. 5,538,848, the content of all of the preceding are hereby incorporated herein by reference in their entirety. Fluorescent analyte detection dyes are known to those of skill in the art. The fluorescent analyte detection dye can be a single fluorescer or a donor-receptor dye pair that is activated by energy transfer in the detection system and can be synthetic or a naturally occurring fluorescer. Appropriate fluorescent analyte detection dyes can be selected for a particular assay and used in accordance with the present invention by those of skill in the art with reference to this disclosure. Known fluorescent dyes useful for labeling oligonucleotides include hydrophobic and stable dyes such as those known by the designations IR792 ([2-[2-[3-[(l,3-dihydro- 3,3-dimethyl-l-propyl-2H-indol-2-ylidine)ethylidine]-2-(phenylthio)-l-cyclohexen-l- yl]ethenyl]-3,3-dimethyl-l-proρylindolium perchlorate]), IR768 ([2-[2-[3-[(l,3-dihydro- 3 , 3 -dimethyl- 1 -propyl-2H-indol-2-ylidine)ethylidine]-2-phenoxy- 1 -cyclohexen- 1 - yl]ethenyl]-3,3-dimethyl-l-propylindolium perchlorate])YL22 ([2-[2-[3-[(l,3-dihydro- 3,3-dimethyl-l-propyl-2H-benzoindol-2-ylidine)ethylidine]-2-(phenylthio)-l-cyclohexen- l-yl]ethenyl]-3,3-dimethyl-l-proρylbenzoindolium iodide]), IR780 ([2-[2-[2-chloro-3- [(1 ,3-dihydro-3 ,3 -dimethyl- 1 -propyl-2H-indol-2-ylidine)ethylidine]- 1 -cyclohexen- 1 - yl]ethenyl]-3,3-dimethyl-l-ρropylindolium perchlorate]), JM5488-48 ([2-[2-[2-chloro-3- [(l,3-dihydro-3,3-dimethyl-l-decanyl-2H-benzoindol-2-ylidine)ethylidine]-l-cyclohexen- l-yl]ethenyl]-3,3-dimethyl-l-decanylbenzoindolium iodide]), JM5488-72 ([2-[2-[-3-[(l,3- dihydro-3 ,3 -dimethyl-1 -decanyl-2H-benzomdol-2-ylidine)ethylidine]- 1 -cyclohexen- 1 - yl]ethenyl]-3,3-dimethyl-l-decanylbenzoindolium iodide]) IR140 and IR143 and their derivatives. Prefened dye structures that can be associated with capture oligonucleotides include IR792, YL22, and the lipophilic forms of cyanine dyes known under the
designations Cy7 (e.g. DiIC18 (7) from Molecular Probes, Inc.) and dibenzoCy7, Cy5, and dibenzoCy5. Further examples of useful fluorescent dyes for labeling individual types of oligonucleotides are known in the art. See, for example, R. P. Haugland, Handbook of Fluorescent Probes and Research Chemicals, 8th Edition, Molecular Probes Inc., Eugene, 1999. For a description of the cyanine dyes Cy5, dibenzoCy5, Cy7, and dibenzoCy7, see Chen, U.S. Patent No. 5,863,401 and Shen et al, U.S. Patent No. 6,002,003, the entire contents of which are hereby incorporated by reference in their entireties. Fluorescing cyanine dyes are commercially available. Light energy selected so as to excite the fluorescent label used in the assay is used to illuminate the capture oligonuleotides bound to the solid substrate, to thereby generate a fluorescent signal. A light source such as a laser or an arc lamp may be used. Preferably, an excitation laser is used to excite the fluorescent labels. Appropriate laser line filters, beam expanders, minors, and lenses may also be used, as will be understood by those with skill in the art with reference to this disclosure. Appropriate lower wavelength lasers for excitation of the analyte dyes are known to those skilled in the art. A prefened excitation wavelength for the fluorescent analyte detection dyes is a 532 nm diode laser, alternatively, a 635 nm diode laser, a 650 nm diode laser, or a 633 nm helium-neon laser can be used. Alternatively, a lower wavelength 488 nm argon-ion, or a 532 nm doubled YAG laser can be used. Longer wavelength lasers (e.g., greater than 750 nm) are known to those skilled in the art. A prefened longer laser excitation wavelength is about 785 nm. Appropriate detectors for detecting a particular emitting light are known as will be understood by those with skill in the art with reference to this disclosure. The detectors can be photodiodes or photomultipliers or similar devices that convert light signals into electrical impulses thereby associating the detected light with its fluorescent source. The detection subsystem can also employ a system of filters, minors, as will be understood by those with skill in the art with reference to this disclosure. The electrical signals from the detectors are typically fed into the electronics of the system for signal and display processing, storage, and/or further processing. Alternatively, light signals may be detected and recorded using a charge coupled device ("CCD") camera, as will be appreciated by one of skill in the art. Hardware, such as a microprocessor in combination with memory storage such as a hard drive in a computer, may be used to collect detected data and to process the data. Suitable hardware used in the analysis system is known as will be understood by those with skill in the art with reference to this disclosure. The analysis system software, used for data and signal processing, can conelate detected data with known data to produce
analytical results. The analysis subsystem can collect data from the electrical signals associated with each specific location on the solid substrate. The processed data and interpreted results can be given as output to a user. A method of using solid substrate arrays to detect target nucleic acids is shown in FIG. 3. A solid substrate having a set of capture nucleotides specific for target nucleic acids deposited on the solid surface in a previously determined array is selected, box 52. The capture oligonucleotides are then exposed to the sample and necessary reagents, box 54. Following exposure, the capture oligonucleotides, and any cleaved assay specific probe hybridized thereto, are processed in an examination zone. The capture oligonucleotides are processed by illuminating the capture oligonucleotides, generating emitted fluorescent light and detecting the emitted light, box 56. The detected emitted light data is correlated with the baseline data to determine the detection of one or more target nucleic acids, box 58. According to the method of the present invention, an analytical sample is allowed to react with a set of oligonucleotides, which are specific to various analytes of interest and which are bound in a predetermined anangement to a solid substrate. The solid substrate is then illuminated such that capture oligonucleotides that have reacted with their specific analyte of interest generate a fluorescent signal. A detection device identifies the location of the emitted signal and conelates this information with the information about the location of the analyte specific capture oligonucleotide to allow quantitative identification of multiple analytes in one reaction. Detection of SNPs In one embodiment, the method of the present invention is used to detect single nucleotide polymorphisms (SNPs) in nucleic acids, as shown in FIG. 4. The target of interest in the target nucleic acid 10 is a specific SNP 60; in this example, the SNP contains a thymine (T). In order to identify the specific SNP, a set 62 of four different types of primary cleavage oligonucleotides are added to the target nucleic acid. All of these primary cleavage oligonucleotides contain a 3' portion 34, which is at least partially complementary to the target nucleic acid immediately adjacent to and upstream of the site of the SNP, and which terminates at the 5' end with one of four nucleotides, containing either adenine (A, 64), cytosine (C, 66), guanine (G, 68) or thymine (T, 70). In addition, each primary cleavage oligonucleotide contains a 5' portion containing a SNP bar code sequence (65, 67, 69, 71) unique for the particular SNP. Thus, for example, the primary cleavage oligonucleotide having an A (64) has a bar code sequence 65 (the "A" bar code sequence) which serves to distinguish it from the other primary cleavage oligonucleotides. When the 3' portion 34 of the primary cleavage oligonucleotide hybridizes with the target nucleic acid, only one of the four sets of primary cleavage oligonucleotides, in
this case the set containing an A, will also hybridize at the site of the SNP to form a primary cleavage structure 72 upon addition of invading oligonucleotide 14. Thus, in the example illustrated in FIG. 4, following addition of a nucleic acid cleavage means, the resulting cleaved assay specific probe 16 will contain an A at its 3' end. Four sets of capture oligonucleotides, each set containing one of four nucleotides A, C, G, or T positioned between a fluor 20 and a quencher 22 at the 5'end, are bound to a solid phase in an array 74 with the location of each set of capture oligonucleotides mapped. Each capture oligonucleotide also contains the complement to an SNP bar code sequence in its 3' portion; for example, a capture oligonucleotide containing a T between the fluor and the quencher (a "T" capture oligonucleotide) is designed so as to have sequence complementary to the "A" SNP bar code in its 3' portion. The capture oligonucleotides are exposed to the cleaved assay specific probe, which hybridizes to the capture oligonucleotides. In the example shown in FIG. 4, the cleaved assay specific probe contains an A at its 3' end and an "A" bar code sequence in its 5' end. Thus, it hybridizes to form a secondary cleavage structure only with those capture oligonucleotides having its complementary sequence, in this case a "T" capture oligonucleotide. Following addition of a nucleic acid cleavage means, the quencher is cleaved off the secondary cleavage structure, resulting in the emission of fluorescent light. The fluorescent light is detected and its position conelated with the predetermined map locations of the capture oligonucleotides to thereby identify the SNP in the target nucleic acid. In FIG. 4, the fluor remains covalently bound to the capture oligonucleotide, and thus to the solid support, following cleavage of the secondary cleavage structure. As will be appreciated, this maintains the fluorescent signal at the predetermined map location in an array to facilitate detection and correlation of the signal to a specific location. It should be noted, however, that the position of the fluor and the quencher on the capture oligonucleotide may be reversed, as shown in FIG. 4 (78). In this embodiment, the primary cleavage reaction is performed in a separate reaction mix so as to allow accumulation of the cleaved assay specific probe. This reaction is then terminated, as by denaturing the nucleic acid cleavage means, and the contents of the reaction transfened to the immobilized capture oligonucleotides. Upon addition of fresh nucleic acid cleavage means, the fluor is released into solution. Rapid image acquisition, as with a CCD camera, would allow signal capture in advance of signal diffusion throughout the bulk solution, so that the location of the signal may be mapped to the predetermined positions of the capture oligonucleotides. Detection of SNPs and Gene Loci In another embodiment of the present invention, multiple genetic loci can be
screened for SNPs in multiplexed assays using multiple bar codes, as shown in FIGs. 5 and 6. Thus, for example, primary cleavage oligonucleotides are designed to include both an SNP bar code and a locus bar code. In FIG. 5, for example, two first cleavage nucleotides 80, 82 are shown, each of which contains the SNP bar code for A (84). However, one first cleavage nucleotide 80 contains a loci bar code (86) for loci 1, while the other 82 contains a loci bar code (88) for loci 2. As is apparent, these two primary cleavage oligonucleotides 80, 82 may be used in the present invention to identify "A" SNPs at gene loci 1 and 2, respectively. Similarly, in FIG. 6, two primary cleavage oligonucleotides 90, 92 are shown for the same gene loci, in this case, loci 1. Each oligonucleotide contains the loci 1 bar code 86, however, one primary cleavage oligonucleotide contains an SNP bar code for G (94) while the other contains an SNP bar code for T (96). As is apparent, these two primary cleavage oligonucleotides 90, 92 may be used in the present invention to identify a "G" SNP or a "T" SNP, respectively, at gene loci 1. As will be appreciated, each gene loci of interest requires four primary cleavage oligonucleotides, each with the appropriate SNP bar code, for the four nucleotides potentially found at the site of the polymorphism, i.e., A, C, G, and T. Alternatively, rather than assigning separate bar codes to each loci and to each SNP, unique primary cleavage oligonucleotides can be designed so that the entire sequence of the 5' portion of the oligonucleotide serves as an indicator of both SNP and gene locus. Thus, each locus would require four unique primary cleavage oligonucleotides conesponding to the SNP identity (A, C, G, T) and the locus identity. In this manner, four hundred unique hairpin capture oligonucleotides would be needed to map one hundred SNPs by perfect match hybridization to the four hundred unique cleaved assay specific probes. Detection of SNPs and Gene Loci using biotin-labeled probes and ELF signal amplification. In another embodiment of the present invention, primary cleavage oligonucleotides are labeled with biotin and cleavage products are detected by ELF signal amplification following hybridization with capture oligonucleotides affixed to a solid substrate, as shown in FIG. 7. In this embodiment, a set 83 of four unique primary cleavage oligonucleotides are provided for each loci of interest in the target nucleic acid 10, each of which is designed to include both an SNP barcode and a loci barcode in the 3' portion, as described above. The 3' end of each primary cleavage oligonucleotide is labeled with biotin 100. The primary cleavage oligonucleotides are hybridized to target nucleic acid in the presence of invading oligonucleotides and nucleic acid cleavage means as described above, and following termination of the cleavage reaction, as by, for example,
denaturation of the nucleic acid cleavage means, the resulting cleaved assay specific probe 102 is recovered. Capture oligonucleotides are designed so as to be complementary to the various cleaved assay specific probes that may be generated in the primary cleavage reaction. These capture oligonucleotides are deposited onto a solid substrate in predetermined locations as described above. As each capture oligonucleotide will contain sequence information specific for a particular gene locus and a particular SNP, each SNP at each locus of interest may be mapped to a specific site in the array. Cleaved assay specific probe from the cleavage reaction is transfened to the solid substrate and allowed to hybridize to capture oligonucleotides containing complementary sequence. Since each locus and SNP may be identified by its specific location in the array, only a single reporter label is required. Thus, for example, where the cleaved assay specific probe is labeled with biotin, Enzyme-Labeled Fluorescence (ELF) signal amplification (Molecular Probes, Eugene, Oregon) may be used to detect hybridization to capture oligonucleotides via CCD camera imaging and digital quantitation, and thus to identify the target nucleic acid of interest, as shown in FIG. 8. Other signal detection systems may also be used, including detection by chemiluminescence. Similarly, other labels than biotin may be employed, including fluorescein isothiocyanate (FITC) or Cy- series near infrared dyes such as Cy3 or Cy5. As will be appreciated, this embodiment allows for high-throughput screening while eliminating the need for hairpin capture oligonucleotides, labeled capture oligonucleotides, or multiple labels for multiple targets. Dual-Labeled Probe In some cases, it may be desirable to remove uncleaved primary cleavage oligonucleotide prior to exposure to capture oligonucleotides. In one embodiment of the present invention, a method is provided wherein dual-labeled primary cleavage oligonucleotides are used. In this embodiment, a detectable label, as for example, FITC, is incorporated at the 5' end of the primary cleavage oligonucleotide, while an affinity tag, such as biotin, is incorporated at the 3' end. Following termination of the cleavage reaction, the assay mix is contacted with a compound having affinity for the affinity tag, such as sfreptavidin in the case of biotin. Optionally, for example, sfreptavidin coated beads may be used to sequester any fragments containing biotin. In one embodiment, sfreptavidin coated beads are used in a device such as the bio-tip described in U.S. Patent No. 5,437,979. Alternatively, magnets may be used to remove sfreptavidin coated magnetic or paramagnetic beads. By first sequestering reactants and products containing the 3' label, including uncleaved primary cleavage oligonucleotide, cleaved assay specific probes containing
only the 5' label may be used for hybridization with capture oligonucleotides without competition from uncleaved reactants. Dual-labeled primary cleavage oligonucleotides are also useful for monitoring the specific activity of nucleic acid cleavage means, such as Cleavase enzymes, as shown in FIG. 9. In this assay, the primary cleavage oligonucleotide is labeled at each end using distinguishable reporters. Two sets of capture oligonucleotides are designed, one set 110 complementary to the 5' end of the primary cleavage oligonucleotide (FLAP) 112, conesponding to the cleaved assay specific probe, and the other set 114 complementary to the 3' end 116. Following the cleavage reaction, reactants are contacted to the capture oligonucleotides and the signal intensities of the captured oligonucleotides are determined. Enzyme specific activity may be determined using the ratio of 5'-label only (conesponding to the cleaved assay specific probe 118) to 5'-label plus 3' label (conesponding to uncleaved primary cleavage oligonucleotide 120). This allows for an estimation of initial velocity and end-points of the reaction. In an alternative embodiment, the primary cleavage oligonucleotide can be internally labeled. Reagent Sets The present invention also contemplates kits and reagent sets useful for practice of the methods described herein. Such kits or reagent sets may include, for example, primary cleavage oligonucleotides, invading oligonucleotides, and capture oligonucleotides deposited onto solid substrates specific for target nucleic acids of interest, control target nucleic acids, nucleic acid cleavage means, various labels and detection means, and instructions for practice of the invention. Having generally described the invention, the same will be more readily understood through reference to the following examples, which are provided by way of illustration, and are not intended to be limiting of the present invention.
Example 1: Cleavage Reaction A reaction mixture was prepared as follows: Invading oligonucleotide (1 μL of 0.5 μM stock solution) was mixed with 4 μL of PEG (16% v/v in 50 mM MOPS, pH 7.5) and the solution placed in a 0.2 L Eppendorf tube. Next, 10 μL of DNA sample target (50 attomoles) was added per tube and overlayed with 20 μL of mineral oil. The invading oligonucleotide-target mixture was incubated for 5 minutes at 95°C to denature double stranded targets. The temperature was reduced to 59°C and 5 μL of Cleavase enzyme (Third Wave) mixture (2 μL, 75mM MgCl2 + 1 μL Cleavase, 100 ng + 1 μL dual-labeled (5'-FITC, 3'-biotin) primary cleavage oligonucleotide, 377 nM + 1 μL distilled water)
added to the tube. The reaction was allowed to proceed 4 hours at 59°C.
Example 2: Cleavage Product Processing The reaction mixture from Example 1 was cooled to 25° C and processed to remove biotinylated primary cleavage oligonucleotide fragment and residual cleavage oligonucleotide from the FITC-labeled cleaved assay specific probe. The reaction mixture (20 μL) was mixed with 30 μL of streptavidin-coated magnetic beads (Calbiochem) and the solution vortexed. The solution was maintained for 1 hour using an end-over-end rotator. Beads were removed from solution by placing the tube next to a magnetic strip for 1-2 minutes.
Example 3: Capture Oligonucleotide Plate Preparation The 5 '-amino terminated capture oligonucleotides complementary to the cleaved assay specific probe were immobilized in the bottom of the wells of an acyl fluoride (ACF) activated polypropylene microplate. Essentially, 10 nL of 10 μM amino capture oligonucleotide prepared in 50 mM sodium carbonate buffer, pH 9, containing 50% v/v MSS (MicroSpotting Solution, Telechem) was deposited on the bottom of the well by contact printing. A Biomek 2000 equipped with an HDRT pin system (Beckman Coulter) was used to deliver the oligonucleotide droplets to the well surface. Following covalent immobilization, the wells were incubated with reagent grade ammonium hydroxide (30% v/v water) for 15 minutes to remove unbound oligonucleotide and cap residual acyl fluoride groups. The wells were then rinsed with water, dried and stored sealed under argon at -20 °C until required for the hybridization experiments.
Example 4: Solid-Phase Hybridization The supernatant containing the cleaved assay specific probes (50 μL) from Example 2 was mixed with 30 μL of SSC Hybridization Buffer containing SDS to bring the final concentration of hybridization buffer to 6X SSC, 0.01% SDS. The 80 μL hybridization mixture was applied to the well of the hybridization plate containing complementary capture oligonucleotides immobilized to the bottom of the well from Example 3. The hybridization was allowed to proceed for 1 hour at 25° C in a humidified shaker-water bath. The solution was removed from the wells and each well rinsed 3 -times with 2X SSC, 0.02%) Tween. An anti-FITC antibody-alkaline phosphatase conjugate (Sigma- Aldrich) was prepared in rinse buffer at 1:1000 v/v and 50 μL applied to each well. The conjugate was incubated for 1 hour at 25° C in a humidified shaker-water bath and processed as previously described. ELF reagent (Molecular Probes) was prepared at 1:100 v/v and 50 μL applied to each well for 30 minutes. After a brief buffer (SSC-
Tween) rinse, the signal was detected using a CCD camera with fluorescent light excitation at 365 nm with lens filtering at emission 520nm. Examples 1-4 are summarized schematically in FIG. 10. In FIG. 10, a primary cleavage structure is formed from target nucleic acid (130) with invading oligonucleotide (132) and primary cleavage oligonucleotide (134). The primary cleavage oligonucleotide is dual-labeled with 5' FITC and 3' biotin. The action of the nucleic acid Cleavase enzyme (136) results in cleavage of the primary cleavage oligonucleotide (134) into the cleaved assay specific fragment (138) and the cleaved primary cleavage oligonucleotide fragment (140). The biotinylated primary cleavage oligonucleotide fragment (140) and remaining primary cleavage oligonucleotide (134) are captured by streptavidin- immobilized magnetic beads (142) which are in turn sequestered or removed from the solution by magnet (144). The FITC-labeled cleaved assay specific fragment (138) is hybridized from solution using the complementary capture oligonucleotide (146) which is immobilized onto solid-support (148). After appropriate rinsing, an anti-FITC antibody- reporter enzyme conjugate (150) is added to the solid-support (148) and signal developed.
Example 5. Effect of Varying the Concentration of Primary Cleavage Oligonucleotide on Removal of Uncleaved Primary Cleavage Oligonucleotide. Primary cleavage structure was prepared with variation in the amount of dual- labeled primary cleavage oligonucleotide (from 37.7 to 377 finoles) and allowed to undergo cleavase treatment for 4 hours at 59 °C as described in Example 1. The temperature was reduced to 25 °C and the reaction mixture incubated with streptavidin- immobilized magnetic beads for 1 hour at 25 °C. After removal of the beads, the supernatant was prepared in hybridization buffer and incubated in wells of the oligonucleotide capture array plate for an additional 1 hour. An anti-FITC antibody-alkaline phosphatase conjugate was then applied to each well for 1 hour. Following rinsing of the wells the ELF reagent was added for 1 hour to generate signal that was subsequently detected using a CCD camera system. FITC-labeled cleaved assay specific probe was detected at all levels of dual labeled primary probe indicating the presence of the SNP within the target. Magnetic bead scavenge of the biotin fragment or uncleaved dual labeled primary cleavage oligonucleotide was most efficient when using 37.7 to 151 finoles dual labeled primary cleavage oligonucleotide, as shown in FIG. 11.
Example 6: Dual Labeled Primary Cleavage Oligonucleotide. In this Example, the assay described above in Examples 1-4 was performed using a dual labeled primary cleavage oligonucleotide, and the effectiveness of scavenging
uncleaved product assessed. Biotin-label detection was performed using sfreptavidin alkaline phosphatase. FITC-label detection was determined using an anti-FITC alkaline phosphatase conjugate. Both signals were developed using the ELF reagent with CCD camera imaging and digital quantitation. The results are shown in FIG. 12. In (A,B) the wild type primary cleavage oligonucleotides were labeled with ST-uotin. Following the procedure set forth in Examples 1-4, the reaction mixture was either used directly for hybridization to the anti-cleaved assay specific probe capture oligonucleotide (A) or first exposed to sfreptavidin coated magnetic beads in order to sequester 5' biotin labeled oligonucleotides (B). In A, substantial capture of both the full (uncleaved) primary cleavage oligonucleotide and the cleaved assay specific probe are indicated, while in B the biotin-labeled probes are efficiently captured by beads resulting in no capture by anti-cleaved assay specific probe capture oligonucleotides. Using a dual-labeled primary cleavage oligonucleotide (FDLPB) in which biotin is placed on the 3' end and the assay-specific portion is labeled with 5' FITC, the relative binding of cleaved assay specific probe and full primary cleavage oligonucleotide can be measured. In C (plus beads), the captured species is primarily the cleaved 5' FITC-labeled assay specific oligonucleotide, while in the absence of beads (D) the full primary cleavage oligonucleotide competes with the cleavage product for anti-cleaved assay specific probe hybridization. As shown in FIG. 12, the dual labeled FDLPB is effectively scavenged by magnetic bead capture, while the cleavage product (5' FITC labeled cleaved assay specific probe) is quantitatively captured by the anti- cleaved assay specific probe capture probe.
While this invention has been described in detail with reference to a certain prefened embodiments, it should be appreciated that the present invention is not limited to those precise embodiments. Rather, in view of the present disclosure which describes the current best mode for practicing the invention, many modifications and variations would present themselves to those of skill in the art without departing from the scope and spirit of this invention. Moreover, this invention is not limited to the particular methodology, protocols, constructs, and reagents described as such may vary, as will be appreciated by one of skill in the art. The scope of the invention is, therefore, indicated by the following claims rather than by the foregoing description. All changes, modifications, and variations coming within the meaning and range of equivalency of the claims are to be considered within their scope. All features disclosed in the specification, including the claims, abstracts, and drawings, and all the steps in any method or process disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are
mutually exclusive. Each feature disclosed in the specification, including the claims, abstract, and drawings, can be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features. Any element in a claim that does not explicitly state "means" for performing a specified function or "step" for performing a specified function, should not be interpreted as a "means" or "step" clause as specified in 35 U.S.C. § 112.
Claims
WHAT IS CLAIMED IS: 1. A method for detecting the presence of a target nucleic acid comprising: (a) providing: (i) a sample comprising at least one target nucleic acid having a first portion, a second portion, and a third portion, wherein the first portion is downstream of and contiguous to the second portion, the second portion is downstream of and contiguous to the third portion, and wherein each target nucleic acid is associated with a preselected bar code sequence; (ii) an invading oligonucleotide, having a 3' portion and a 5' portion, the 5' portion containing sequence complementary to the first portion of the target nucleic acid and the 3' portion containing sequence complementary to the second portion of the target nucleic acid; (iii) a primary cleavage oligonucleotide, each primary cleavage oligonucleotide having a 3' portion complementary to the third portion of the target nucleic acid, a cleavage portion complementary to the second portion of the target nucleic acid, and a 5' portion having a bar code sequence specific for the target nucleic acid; (iv) a nucleic acid cleavage means; (b) generating a primary cleavage structure for each target nucleic acid, wherein at least the 3' portion of the primary cleavage oligonucleotide is annealed to a conesponding target nucleic acid and at least the 5' portion of the invading oligonucleotide is annealed to a corresponding target nucleic acid, wherein cleavage of the primary cleavage structure is performed by the nucleic acid cleavage means to cleave each primary cleavage oligonucleotide to generate a cleaved assay specific probe conesponding to each target nucleic acid, each cleaved assay specific probe having a 5' region and a 3' region; (c) providing a capture oligonucleotide for each target nucleic acid, each capture oligonucleotide having contiguous first and second portions, wherein the first portion comprises a sequence complementary to the bar code sequence associated with the target nucleic acid and a 3' end of the capture oligonucleotide, the 3' end of the capture oligonucleotide is coupled to a solid support in a predetermined location, such that each capture oligonucleotide bar code sequence is associated with a specific site on the solid support, wherein the second portion comprises a 5' end of the capture oligonucleotide, a region of self-complementarity, and is coupled to a detectable label in a first state, wherein the first portion of each capture oligonucleotide is complementary to a cleaved assay specific probe for a conesponding target nucleic acid, and wherein a part of the second portion of each capture oligonucleotide is complementary to at least a portion of the 3' region of the conesponding cleaved assay specific probe; (d) generating a secondary cleavage structure, wherein the 5' end of each capture oligonucleotide is cleaved from the remainder of the second region of each capture oligonucleotide changing the detectable label to a second state; (e) detecting the cleavage of the capture oligonucleotides by a change in fluorescence; and (f) associating the detected change in fluorescence with a specific site on the solid substrate thereby identifying the bar code sequence associated with the change in fluorescence and detecting the target nucleic acid corresponding to the bar code sequence.
2. The method of claim 1, wherein the solid support coupled to the 3' end of each capture oligonucleotide comprises an activated microplate.
3. The method of claim 1, wherein the detectable label coupled to the capture oligonucleotide is a fluorescent analyte detection dye, the analyte detection dye being excitable by light at an excitation wavelength and capable of emitting light at a maximum wavelength when excited.
4. The method of claim 3, wherein the fluorescent analyte detection dye comprises a fluorescent reporter and a quencher molecule, the quencher molecule being removed by cleavage of the capture oligonucleotide.
5. The method of claim 1, wherein the method is used to detect single nucleotide polymorphisms.
6. The method of claim 1, wherein the method is used to detect specific gene loci.
7. The method of claim 6, wherein the method is further used to detect a single nucleotide polymorphisms.
8. The method of claim 1, wherein the nucleic acid cleavage means comprises a structure-specific nuclease.
9. The method of claim 1, wherein the nucleic acid cleavage means comprises a thermostable 5' nuclease.
10. The method of claim 1, wherein the target nucleic acid comprises single stranded DNA.
11. The method of claim 1, wherein the target nucleic acid comprises double stranded DNA.
12. The method of claim 1 , wherein the target nucleic acid comprises RNA.
13. A method for detecting and identifying a single nucleotide polymorphism (SNP) in a target nucleic acid molecule in an assay, comprising: (a) providing: (i) at least one target nucleic acid molecule having a first portion, a second portion comprising a SNP, and a third portion, wherein the first portion is downsfream of and continguous to the second portion, the second portion is downstream of and contiguous to the third portion; (ii) an invading oligonucleotide, having a 3' portion and a 5' portion, the 5' portion of the invading oligonucleotide containing sequence complementary to the first portion of the target nucleic; (iii) a set of primary cleavage oligonucleotides, each primary cleavage oligonucleotide having a 3' portion complementary to the third portion of the target nucleic acid, a middle portion containing a nucleotide having a base selected from the group consisting of A, T, C, and G, and a 5' portion containing a SNP-specific bar code sequence; (iv) a nucleic acid cleavage means; (b) mixing the target nucleic acid, the invading oligonucleotides, the set of primary cleavage oligonucleotides and the nucleic acid cleavage means to create a reaction mixture under reaction conditions such that at least the 3' portion and middle portion of those primary cleavage oligonucleotides having a middle portion complementary to the SNP portion of the target nucleic acid are annealed to the target nucleic acid and wherein at least the 5' portion of the invading oligonucleotide is amiealed to the target nucleic acid so as to create a primary cleavage structure, wherein cleavage of the primary cleavage structure occurs to generate a cleaved assay specific probe comprising the 5' portion and middle portion of those primary cleavage oligonucleotides having a middle portion complementary to the SNP portion of the target nucleic acid, such that the cleaved assay specific probe terminates at its 3' end with a nucleotide complementary to the SNP portion of the target nucleic acid; (c) providing an array comprising a set of capture oligonucleotides and a solid phase, each capture oligonucleotide having a 5' portion, a middle portion, and a 3' portion, the 3' portion being attached at the 3' end to the solid phase and containing sequence complementary the SNP-specific bar code sequence, the middle portion having a sequence capable of forming a hairpin loop, the middle portion being labeled with a reporter and terminating at its 5' end with a nucleotide containing a base selected from the group consisting of A, C, T, and G, and the 5' portion containing a quencher, such that the quencher interacts with the reporter to prevent the reporter from producing a signal, wherein the bar code sequence of each capture oligonucleotide is associated with a location on the array; (d) adding the cleaved assay specific probe from the reaction mixture to the array to create an array mixture under reaction conditions such that at least the 5' portion of the cleaved assay specific probe is annealed to the 3' portion of the capture oligonucleotide and the nucleotide at the 3' end of the cleaved assay specific probe is annealed to the nucleotide at the 5' end of the middle portion of the capture oligonucleotide so as to create a secondary cleavage structure; (e) adding nucleic acid cleavage means to the array mixture to thereby cleave the second cleavage structure, releasing the quencher from the capture oligonucleotide; (f) detecting signal from the reporter and associating it with its location on the array to identify the bar code sequence contained in the capture oligonucleotides from which the quencher was removed to thereby identify the SNP in the target nucleic acid.
14. The method of claim 13, wherein the nucleic acid cleavage means comprises a structure-specific nuclease.
15. The method of claim 13, wherein the nucleic acid cleavage means comprises a thermostable 5' nuclease.
16. The method of claim 13, wherein the target nucleic acid comprises single stranded DNA.
17. The method of claim 13, wherein the target nucleic acid comprises double stranded DNA.
18. The method of claim 13, wherein the target nucleic acid comprises RNA.
19. The method of claim 13, wherein the reporter is a fluorescent dye.
20. A method for detecting and identifying a single nucleotide polymorphism at a specific gene locus in a target nucleic acid molecule in an assay, comprising: (a) providing: (i) at least one target nucleic acid molecule having a first portion, a second portion comprising a SNP, and a third portion, wherein the first portion is downstream of and continguous to the second portion, the second portion is downstream of and contiguous to the third portion; (ii) an invading oligonucleotide, having a 3' portion and a 5' portion, the 5' portion of the invading oligonucleotide containing sequence complementary to the first portion of the target nucleic; (iii) at least one set of primary cleavage oligonucleotides for each gene locus, each primary cleavage oligonucleotide having a 3' portion complementary to the third portion of the target nucleic acid, a middle portion containing a nucleotide having a base selected from the group consisting of A, T, C, and G, and a 5' portion containing an SNP-specific bar code sequence and a gene locus-specific bar code sequence; (iv) a nucleic acid cleavage means; (b) mixing the target nucleic acid, the invading oligonucleotides, the sets of primary cleavage oligonucleotides and the nucleic acid cleavage means to create a reaction mixture under reaction conditions such that at least the 3' portion and middle portion of those primary cleavage oligonucleotides having a middle portion complementary to the SNP portion of the target nucleic acid and a 3' portion complementary to the gene locus on the target nucleic acid are annealed to the target nucleic acid and wherein at least the 5' portion of the invading oligonucleotide is annealed to the target nucleic acid so as to create a primary cleavage structure, wherein cleavage of the primary cleavage structure occurs to generate a cleaved assay specific probe comprising the 5' portion and middle portion of those primary cleavage oligonucleotides having a middle portion complementary to the SNP portion of the target nucleic acid, such that the cleaved assay specific probe terminates at its 3' end with a nucleotide complementary to the SNP portion of the target nucleic acid; (c) providing an array comprising a set of capture oligonucleotides and a solid phase, each capture oligonucleotide having a 5' portion, a middle portion, and a 3' portion, the 3' portion being attached at the 3' end to the solid phase and containing sequence complementary the SNP-specific bar code sequence and the gene locus bar code sequence, the middle portion having a sequence capable of forming a hairpin loop, the middle portion being labeled with a reporter and terminating at its 5' end with a nucleotide containing a base selected from the group consisting of A, C, T, and G, and the 5' portion containing a quencher, such that the quencher interacts with the reporter to prevent the reporter from producing a signal, wherein the bar code sequences of each capture oligonucleotide are associated with a location on the anay; (d) adding the cleaved assay specific probe from the reaction mixture to the anay to create an array mixture under reaction conditions such that at least the 5' portion of the cleaved assay specific probe is annealed to the 3' portion of the capture oligonucleotide and the nucleotide at the 3' end of the cleaved assay specific probe is annealed to the nucleotide at the 5' end of the middle portion of the capture oligonucleotide so as to create a secondary cleavage structure; (e) adding nucleic acid cleavage means to the array mixture to thereby cleave the second cleavage structure, releasing the quencher from the capture oligonucleotide; (f) detecting signal from the reporter and associating it with its location on the array to identify the bar code sequences contained in the capture oligonucleotides from which the quencher was removed to thereby identify the SNP and gene locus in the target nucleic acid.
21. The method of claim 20, wherein the nucleic acid cleavage means comprises a structure-specific nuclease.
22. The method of claim 20, wherein the nucleic acid cleavage means comprises a thermostable 5' nuclease.
23. The method of claim 20, wherein the target nucleic acid comprises single stranded DNA.
24. The method of claim 20, wherein the target nucleic acid comprises double stranded DNA.
25. The method of claim 20, wherein the target nucleic acid comprises RNA.
26. The method of claim 20, wherein the reporter is a fluorescent dye.
27. A method of detecting and identifying a single nucleotide polymorphism (SNP) in a target nucleic acid molecule in an assay, comprising: (a) providing: (i) a target nucleic acid molecule having a first portion, a second portion comprising a SNP, and a third portion, wherein the first portion is downstream of and contiguous to the second portion, the second portion is downstream of and contiguous to the third portion; (ii) an invading oligonucleotide, having a 3' portion and a 5' portion, the 5' portion of the invading oligonucleotide containing sequence complementary to the first portion of the target nucleic; (iii) a set of primary cleavage oligonucleotides, each primary cleavage oligonucleotide having a 3' portion complementary to the third portion of the target nucleic acid, a middle portion containing a nucleotide containing a base selected from the group consisting of A, T, C, and G, and a 5' portion containing an SNP-specific bar code sequence; (iv) an array comprising a set of capture oligonucleotides and a solid phase, each capture oligonucleotide having a 5' portion, a middle portion, and a 3' portion, the 3' portion being attached at the 3' end to the solid phase and containing sequence complementary the SNP-specific bar code sequence, wherein the bar code sequence of each capture oligonucleotide is associated with a location on the array, the middle portion having a sequence capable of forming a hairpin loop, the middle portion being labeled with a reporter and terminating at its 5' end with a nucleotide containing a base selected from the group consisting of A, C, T, and G, and the 5' portion containing a quencher, such that the quencher interacts with the reporter to prevent the reporter from producing a signal; (v) a nucleic acid cleavage means; (b) mixing the target nucleic acid, the invading oligonucleotides, the primary cleavage oligonucleotides and the nucleic acid cleavage means in the array to create a reaction mixture under reaction conditions such that at least the 3' portion and middle portion of those primary cleavage oligonucleotides having a middle portion complementary to the SNP portion of the target nucleic acid are annealed to the target nucleic acid and wherein at least the 5' portion of the invading oligonucleotide is annealed to the target nucleic acid so as to create a cleavage structure, wherein cleavage of the cleavage structure occurs to generate cleaved assay specific probes comprising the 5' portion and middle portion of those primary cleavage oligonucleotides having a middle portion complementary to the SNP portion of the target nucleic acid, such that the cleaved assay specific probe terminates at its 3' end with a nucleotide complementary to the SNP portion of the target nucleic acid; (c) incubating the reaction mixture under reaction conditions such at least the 5' portion of the cleaved assay specific probe is annealed to the 3' portion of the capture oligonucleotide and the nucleotide at the 3' end of the cleaved assay specific probe is annealed to the nucleotide at the 5' end of the middle portion of the capture oligonucleotide so as to create a secondary cleavage structure, wherein cleavage of the secondary cleavage structure occurs to thereby release the quencher from the capture oligonucleotide; (d) detecting signal from the reporter and associating it with its location on the anay to identify the bar code sequence contained in the capture oligonucleotides from which the quencher was removed to thereby identify the SNP in the target nucleic acid.
28. The method of claim 27, wherein the nucleic acid cleavage means comprises a structure-specific nuclease.
29. The method of claim 27, wherein the nucleic acid cleavage means comprises a thermostable 5' nuclease.
30. The method of claim 27, wherein the target nucleic acid comprises single stranded DNA.
31. The method of claim 27, wherein the target nucleic acid comprises double stranded DNA.
32. The method of claim 27, wherein the target nucleic acid comprises RNA.
33. The method of claim 27, wherein the reporter is a fluorescent dye.
34. A method of detecting and identifying a single nucleotide polymorphism (SNP) and a gene locus in a target nucleic acid molecule in an assay, comprising: (a) providing: (i) a target nucleic acid molecule having a first portion, a second portion comprising a SNP, and a third portion, wherein the first portion is downstream of and contiguous to the second portion, the second portion is downstream of and contiguous to the third portion; (ii) an invading oligonucleotide, having a 3' portion and a 5' portion, the 5' portion of the invading oligonucleotide containing sequence complementary to the first portion of the target nucleic; (iii) at least one set of primary cleavage oligonucleotides for each gene locus, each primary cleavage oligonucleotide having a 3' portion complementary to the third portion of the target nucleic acid, a middle portion containing a nucleotide having a base selected from the group consisting of A, T, C, and G, and a 5' portion containing an SNP-specific bar code sequence and a gene locus-specific bar code sequence; (iv) an array comprising a set of capture oligonucleotides and a solid phase, each capture oligonucleotide having a 5' portion, a middle portion, and a 3' portion, the 3' portion being attached at the 3' end to the solid phase and containing sequences complementary the SNP-specific bar code sequence and the gene locus bar code sequence, wherein the bar code sequences of each capture oligonucleotide is associated with a location on the array, the middle portion having a sequence capable of forming a hairpin loop, the middle portion being labeled with a reporter and terminating at its 5' end with a nucleotide containing a base selected from the group consisting of A, C, T, and G, and the 5' portion containing a quencher, such that the quencher interacts with the reporter to prevent the reporter from producing a signal; (v) a nucleic acid cleavage means; (b) mixing the target nucleic acid, the invading oligonucleotides, the primary cleavage oligonucleotides and the nucleic acid cleavage means in the anay to create a reaction mixture under reaction conditions such that at least the 3' portion and middle portion of those primary cleavage oligonucleotides having a middle portion complementary to the SNP portion of the target nucleic acid are annealed to the target nucleic acid and wherein at least the 5' portion of the invading oligonucleotide is annealed to the target nucleic acid so as to create a cleavage structure, wherein cleavage of the cleavage structure occurs to generate cleaved assay specific probes comprising the 5' portion and middle portion of those primary cleavage oligonucleotides having a middle portion complementary to the SNP portion of the target nucleic acid, such that the cleaved assay specific probe terminates at its 3' end with a nucleotide complementary to the SNP portion of the target nucleic acid; (c) incubating the reaction mixture under reaction conditions such at least the 5' portion of the cleaved assay specific probe is annealed to the 3' portion of the capture oligonucleotide and the nucleotide at the 3' end of the cleaved assay specific probe is annealed to the nucleotide at the 5' end of the middle portion of the capture oligonucleotide so as to create a secondary cleavage structure, wherein cleavage of the secondary cleavage structure occurs to thereby release the quencher from the capture oligonucleotide; (d) detecting signal from the reporter and associating it with its location on the array to identify the bar code sequences contained in the capture oligonucleotides from which the quencher was removed to thereby identify the SNP and gene locus in the target nucleic acid.
35. The method of claim 34, wherein the nucleic acid cleavage means comprises a structure-specific nuclease.
36. The method of claim 34, wherein the nucleic acid cleavage means comprises a thermostable 5' nuclease.
37. The method of claim 34, wherein the target nucleic acid comprises single stranded DNA.
38. The method of claim 34, wherein the target nucleic acid comprises double stranded DNA.
39. The method of claim 34, wherein the target nucleic acid comprises RNA.
40. The method of claim 35, wherein the reporter is a fluorescent dye.
41. A method of detecting and identifying a single nucleotide polymorphism (SNP) at a specific locus in a target nucleic acid molecule in an assay, comprising: (a) providing: (i) a target nucleic acid molecule having a first portion, a second portion comprising a SNP, and a third portion, wherein the first portion is downstream of and contiguous to the second portion, the second portion is downsfream of and contiguous to the third portion; (ii) an invading oligonucleotide having a 3' portion and a 5' portion, the 5' portion of the invading oligonucleotide containing sequence complementary to the first portion of the target nucleic; (iii) at least one set of primary cleavage oligonucleotides, each primary cleavage oligonucleotide having a 3' portion complementary to the third portion of the target nucleic acid, a middle portion containing a nucleotide having a base selected from the group consisting of A, T, C, and G, and a 5' portion containing a SNP-specific bar code sequence and a locus-specific bar code sequence; (iv) a nucleic acid cleavage means; (b) mixing the target nucleic acid, the invading oligonucleotides, the primary cleavage oligonucleotides and the nucleic acid cleavage means to create a reaction mixture under reaction conditions such at least the 3' portion and middle portion of those primary cleavage oligonucleotides having a middle portion complementary to the SNP portion of the target nucleic acid are annealed to the target nucleic acid and wherein at least the 5' portion of the invading oligonucleotide is amiealed to the target nucleic acid so as to create a cleavage structure, wherein cleavage of the cleavage structure occurs to generate cleavage products comprising the 5' portion and middle portion of those primary cleavage oligonucleotides having a middle portion complementary to the SNP portion of the target nucleic acid; and (c) detecting the SNP-specific sequence and the locus-specific sequence in the cleavage products to thereby identify the SNP and the locus in the target nucleic acid.
42. The method of claim 41, wherein the nucleic acid cleavage means comprises a structure-specific nuclease.
43. The method of claim 41, wherein the nucleic acid cleavage means comprises a thermostable 5' nuclease.
44. The method of claim 41, wherein the target nucleic acid comprises single stranded DNA.
45. The method of claim 41, wherein the target nucleic acid comprises double stranded DNA.
46. The method of claim 41, wherein the target nucleic acid comprises RNA.
47. A method of detecting and identifying a single nucleotide polymorphism (SNP) at a specific locus in a target nucleic acid molecule in an assay, comprising: (a) providing: (i) a target nucleic acid molecule having a first portion, a second portion comprising a SNP, and a third portion, wherein the first portion is downstream of the second portion, the second portion is downstream of and contiguous to the third portion; (ii) an invading oligonucleotide having a 3' portion and a 5' portion, the 5' portion of the invading oligonucleotide containing sequence complementary to the first portion of the target nucleic; (iii) a set of primary cleavage oligonucleotides comprising four unique oligonucleotides for each locus of interest, such that each primary cleavage oligonucleotide contains a unique sequence ("bar code") denoting a specific locus and a specific SNP, each primary cleavage oligonucleotide having a 3' portion complementary to the third portion of the target nucleic acid, a middle portion containing a nucleotide selected from the group consisting of A, T, C, and G, and a 5' portion containing the bar code; (iv) a nucleic acid cleavage means; (b) mixing the target nucleic acid, the invading oligonucleotides, the primary cleavage oligonucleotides and the nucleic acid cleavage means to create a reaction mixture under reaction conditions such at least the 3' portion and middle portion of those primary cleavage oligonucleotides having a middle portion complementary to the SNP portion of the target nucleic acid are annealed to the target nucleic acid and wherein at least the 5' portion of the invading oligonucleotide is annealed to the target nucleic acid so as to create a cleavage structure, wherein cleavage of the cleavage structure occurs to generate cleaved assay specific probes comprising the 5' portion and middle portion of those primary cleavage oligonucleotides having a middle portion complementary to the SNP portion of the target nucleic acid; (c) providing an array comprising a set of capture oligonucleotides and a solid phase, each capture oligonucleotide having a 5' portion, a middle portion, and a 3' portion, the 3' portion being attached at the 3' end to the solid phase and containing sequence complementary to the bar code, the middle portion having a sequence capable of forming a hairpin loop, the middle portion being labeled with a reporter and terminating at its 5' end with a nucleotide selected from the group consisting of A, C, T, and G, and the 5' portion contain a quencher, such that the quencher interacts with the reporter to prevent the reporter from producing a signal; (d) adding the cleaved assay specific probe from the reaction mixture to the array to create an anay mixture under reaction conditions such at least the 5' portion of the cleaved assay specific probe is annealed to the 3' portion of the capture oligonucleotide and the nucleotide at the 3' end of the cleavage product is annealed to the nucleotide at the 5' end of the middle portion of the capture oligonucleotide so as to create a secondary cleavage structure, wherein cleavage of the secondary cleavage structure occurs to release the quencher from the capture oligonucleotide; (e) detecting signal from the reporter to identify those capture oligonucleotides from which the quencher was removed to thereby identify the SNP and locus in the target nucleic acid.
48. The method of claim 47, wherein the nucleic acid cleavage means comprises a structure-specific nuclease.
49. The method of claim 47, wherein the nucleic acid cleavage means comprises a thermostable 5' nuclease.
50. The method of claim 47, wherein the target nucleic acid comprises single stranded DNA.
51. The method of claim 47, wherein the target nucleic acid comprises double stranded DNA.
52. The method of claim 47, wherein the target nucleic acid comprises RNA.
53. The method of claim 47, wherein the reporter is a fluorescent dye.
54. A method of detecting and identifying a single nucleotide polymorphism (SNP) at a specific locus in a target nucleic acid molecule in an assay, comprising: (a) providing: (i) a target nucleic acid molecule having a first portion, a second portion comprising an SNP, and a third portion, wherein the first portion is downsfream of the second portion, the second portion is downstream of and contiguous to the third portion; (ii) an invading oligonucleotide having a 3' portion and a 5' portion, the 5' portion of the invading oligonucleotide containing sequence complementary to the first portion of the target nucleic; (iii) a set of primary cleavage oligonucleotides comprising four unique oligonucleotides for each locus of interest, such that each primary cleavage oligonucleotide contains a unique sequence ("bar code") denoting a specific locus and a specific SNP, each primary cleavage oligonucleotide having a 3' portion complementary to the third portion of the target nucleic acid, a middle portion comprising a nucleotide selected from the group consisting of A, T, C, and G, and a 5' portion containing the bar code and a detectable label; (iv) a nucleic acid cleavage means; (b) mixing the target nucleic acid, the invading oligonucleotides, the primary cleavage oligonucleotides and the nucleic acid cleavage means to create a reaction mixture under reaction conditions such at least the 3' portion and middle portion of those primary cleavage oligonucleotides having a middle portion complementary to the SNP portion of the target nucleic acid are annealed to the target nucleic acid and wherein at least the 5' portion of the invading oligonucleotide is annealed to the target nucleic acid so as to create a cleavage structure, wherein cleavage of the cleavage structure occurs to generate cleaved assay specific probes comprising the 5' portion and middle portion of those primary cleavage oligonucleotides having a middle portion complementary to the SNP portion of the target nucleic acid; and (c) detecting the bar code in the cleaved assay specific probe to thereby identify the SNP and the locus in the target nucleic acid.
55. The method of claim 54, wherein the nucleic acid cleavage means comprises a structure-specific nuclease.
56. The method of claim 54, wherein the nucleic acid cleavage means comprises a thermostable 5' nuclease.
57. The method of claim 54, wherein the target nucleic acid comprises single stranded DNA.
58. The method of claim 54, wherein the target nucleic acid comprises double stranded DNA.
59. The method of claim 54, wherein the target nucleic acid comprises RNA.
60. The method of claim 47, wherein the detectable label is biotin.
61. A method of detecting and identifying a single nucleotide polymorphism (SNP) at a specific locus in a target nucleic acid molecule in an assay, comprising: (a) providing: (i) a target nucleic acid molecule having a first portion, a second portion comprising a SNP, and a third portion, wherein the first portion is downstream of the second portion, the second portion is downstream of and contiguous to the third portion; (ii) an invading oligonucleotide having a 3' portion and a 5' portion, the 5' portion of the invading oligonucleotide containing sequence complementary to the first portion of the target nucleic; (iii) a set of primary cleavage oligonucleotides comprising four unique oligonucleotides for each locus of interest, such that each primary cleavage oligonucleotide contains a unique sequence ("bar code") denoting a specific locus and a specific SNP, each primary cleavage oligonucleotide having a 3' portion complementary to the third portion of the target nucleic acid, a middle portion comprising a nucleotide selected from the group consisting of A, T, C, and G, and a 5' portion containing the bar code and a detectable label; (iv) a nucleic acid cleavage means; (b) mixing the target nucleic acid, the invading oligonucleotides, the primary cleavage oligonucleotides and the nucleic acid cleavage means to create a reaction mixture under reaction conditions such at least the 3' portion and middle portion of those primary cleavage oligonucleotides having a middle portion complementary to the SNP portion of the target nucleic acid are annealed to the target nucleic acid and wherein at least the 5' portion of the invading oligonucleotide is annealed to the target nucleic acid so as to create a cleavage structure, wherein cleavage of the cleavage structure occurs to generate cleaved assay specific probes comprising the 5' portion and middle portion of those primary cleavage oligonucleotides having a middle portion complementary to the SNP portion of the target nucleic acid; (c) providing an array comprising a set of capture oligonucleotides and a solid phase, each capture oligonucleotide being attached at one end to the solid phase and containing sequence complementary to the bar code, wherein the location of each capture oligonucleotide on the solid phase is conelated with the bar code contained in the capture oligonucleotide; (d) adding the cleaved assay specific probe from the reaction mixture to the array to create an anay mixture under reaction conditions such that at least some portion of the cleaved assay specific probe is annealed to those capture oligonucleotides containing sequence complementary to the bar code sequence contained in the cleaved assay specific probe; (e) removing unannealed cleaved assay specific probe; and (f) detecting the label on annealed cleaved assay specific probe, conelating the position of the annealed cleaved assay specific probe on the anay to the bar code of the capture oligonucleotide, to thereby identify the SNP and locus in the target nucleic acid.
62. The method of claim 61, wherein the nucleic acid cleavage means comprises a structure-specific nuclease.
63. The method of claim 61, wherein the nucleic acid cleavage means comprises a thermostable 5' nuclease.
64. The method of claim 61, wherein the target nucleic acid comprises single stranded DNA.
65. The method of claim 61, wherein the target nucleic acid comprises double stranded DNA.
66. The method of claim 61, wherein the target nucleic acid comprises RNA.
67. The method of claim 61 , wherein the detectable label is biotin.
68. The method of claim 61, wherein the primary cleavage oligonucleotide further comprises an affinity label at the 3' end.
69. The method of claim 68, wherein the affinity label is biotin.
70. The method of claim 68, further comprising the step of contacting the reaction mixture to a substrate comprising an agent having affinity for the affinity label, to thereby remove uncleaved primary cleavage oligonucleotide
71. The method of claim 70, wherein the affinity label is biotin and the agent having affinity for the biotin is sfreptavidin.
72. The method of claim 71 , wherein the substrate is a magnetic bead.
73. A method of monitoring the specific activity of a nucleic acid cleavage means, comprising: (a) providing: (i) a target nucleic acid molecule having a first portion, a second portion, and a third portion, wherein the first portion is downstream of the second portion, the second portion is downstream of and contiguous to the third portion; (ii) an invading oligonucleotide having a 3' portion and a 5' portion, the 5' portion of the invading oligonucleotide containing sequence complementary to the first portion of the target nucleic; (iii) a primary cleavage oligonucleotide, each primary cleavage oligonucleotide having a 5' portion containing a first detectable label and a 3' portion complementary to the third portion of the target nucleic acid and containing a second detectable label; (iv) a nucleic acid cleavage means; (b) mixing the target nucleic acid, the invading oligonucleotide, the primary cleavage oligonucleotide and the nucleic acid cleavage means to create a reaction mixture under reaction conditions such that at least the 3' portion of the primary cleavage oligonucleotide is annealed to the target nucleic acid and wherein at least the 5' portion of the invading oligonucleotide is annealed to the target nucleic acid so as to create a cleavage structure, wherein cleavage of the cleavage structure occurs to generate a cleavage product comprising the 5' portion of the primary cleavage oligonucleotide and the first label; (c) denaturing the reaction mixture; (d) providing an anay comprising a capture oligonucleotide and a solid phase, the capture oligonucleotide being attached at one end to the solid phase and containing sequence complementary to the 5' portion of the primary cleavage oligonucleotide; (e) adding the cleavage products from the reaction mixture to the array under reaction conditions such that the cleavage product containing the 5'end and first label as well as uncleaved primary cleavage oligonucleotide containing both the first and the second label is annealed to the capture oligonucleotide, and washing the array to remove unannealed cleavage product; (f) measuring the signal intensity of the first label to thereby provide a measure of the total amount of cleavage product containing the 5'end and first label as well as the uncleaved primary cleavage oligonucleotide containing both the first and the second label; (g) measuring the signal intensity of the second label to thereby provide a measure of the uncleaved primary cleavage oligonucleotide containing both the first and the second label; (h) calculating the amount of cleavage product containing the 5'end and first label and determining the ratio of cleavage product containing the 5'end and first label to cleavage product containing the 5'end and first label plus uncleaved primary cleavage oligonucleotide to thereby provide a measure of the specific acitivity of the nucleic acid cleavage means.
74. A kit for detecting and identifying a single nucleotide polymorphism (SNP) at a specific locus in a target nucleic acid molecule in an assay, the kit comprising: a set of primary cleavage oligonucleotides corresponding to the target nucleic acid comprising four unique oligonucleotides for each locus of interest, such that each primary cleavage oligonucleotide contains a unique sequence ("bar code") denoting a specific locus and a specific SNP, each primary cleavage oligonucleotide having a 3' portion complementary to the target nucleic acid, a middle portion comprising a nucleotide selected from the group consisting of A, T, C, and G, and a 5' portion containing the bar code and a detectable label; an invading oligonucleotide corresponding to the target nucleic acid; a nucleic acid cleavage means for cleaving a primary cleavage structure formed by the primary cleavage oligonucleotide, the invading oligonucleotide and the target nucleic acid to form a cleaved assay specific probe; an array comprising a set of capture oligonucleotides and a solid phase, each capture oligonucleotide being attached at one end to the solid phase and containing sequence complementary to the cleaved assay specific probe, including the bar code, wherein the location of each capture oligonucleotide on the solid phase is correlated with the bar code contained in the capture oligonucleotide.
75. The kit of claim 74 wherein the solid support is an activated microplate and the detectable label is biotin.
76. The kit of claim 74 further comprising a detector for detecting annealing of the cleaved assay specific probe to the capture oligonucleotide.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU2003300271A AU2003300271A1 (en) | 2003-12-19 | 2003-12-19 | Solid-phase multiplexed invader assay |
| PCT/US2003/040973 WO2005068660A1 (en) | 2003-12-19 | 2003-12-19 | Solid-phase multiplexed invader assay |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/US2003/040973 WO2005068660A1 (en) | 2003-12-19 | 2003-12-19 | Solid-phase multiplexed invader assay |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2005068660A1 true WO2005068660A1 (en) | 2005-07-28 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/US2003/040973 WO2005068660A1 (en) | 2003-12-19 | 2003-12-19 | Solid-phase multiplexed invader assay |
Country Status (2)
| Country | Link |
|---|---|
| AU (1) | AU2003300271A1 (en) |
| WO (1) | WO2005068660A1 (en) |
Cited By (7)
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|---|---|---|---|---|
| EP1668157A4 (en) * | 2003-07-07 | 2007-10-24 | One Cell Systems Inc | Hairpin-labeled probes and methods of use |
| WO2008083261A1 (en) * | 2006-12-29 | 2008-07-10 | Applera Corporation | Systems and methods for detecting nucleic acids |
| WO2008083259A1 (en) * | 2006-12-29 | 2008-07-10 | Applera Corporation | Systems and methods for detecting nucleic acids |
| DE102008040513A1 (en) * | 2008-07-17 | 2010-01-21 | BAM Bundesanstalt für Materialforschung und -prüfung | Use of long-wavelength emitting cyanine compound as NIR fluorescence standard and kit for calibration of photoluminescence measurement systems |
| EP2180066A1 (en) * | 2008-10-23 | 2010-04-28 | Honeywell International | Single nucleotide polymorphism genotyping detection via the real-time invader assay microarray platform |
| US20110033846A1 (en) * | 2006-12-13 | 2011-02-10 | Nanibhushan Dattagupta | Concurrent Analysis of Multiple Patient Samples Using Solid Phase Addressable Multiplex Test with High Signal-to-Noise Ratio |
| CN104093857A (en) * | 2011-11-17 | 2014-10-08 | Nvs技术股份有限公司 | Quantitative, highly multiplexed detection of nucleic acids |
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|---|---|---|---|---|
| US5994069A (en) * | 1996-01-24 | 1999-11-30 | Third Wave Technologies, Inc. | Detection of nucleic acids by multiple sequential invasive cleavages |
| US6528254B1 (en) * | 1999-10-29 | 2003-03-04 | Stratagene | Methods for detection of a target nucleic acid sequence |
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2003
- 2003-12-19 WO PCT/US2003/040973 patent/WO2005068660A1/en active Application Filing
- 2003-12-19 AU AU2003300271A patent/AU2003300271A1/en not_active Abandoned
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5994069A (en) * | 1996-01-24 | 1999-11-30 | Third Wave Technologies, Inc. | Detection of nucleic acids by multiple sequential invasive cleavages |
| US6528254B1 (en) * | 1999-10-29 | 2003-03-04 | Stratagene | Methods for detection of a target nucleic acid sequence |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1668157A4 (en) * | 2003-07-07 | 2007-10-24 | One Cell Systems Inc | Hairpin-labeled probes and methods of use |
| US20110033846A1 (en) * | 2006-12-13 | 2011-02-10 | Nanibhushan Dattagupta | Concurrent Analysis of Multiple Patient Samples Using Solid Phase Addressable Multiplex Test with High Signal-to-Noise Ratio |
| US8691508B2 (en) * | 2006-12-13 | 2014-04-08 | Autogenomics, Inc. | Concurrent analysis of multiple patient samples using solid phase addressable multiplex test with high signal-to-noise ratio |
| WO2008083261A1 (en) * | 2006-12-29 | 2008-07-10 | Applera Corporation | Systems and methods for detecting nucleic acids |
| WO2008083259A1 (en) * | 2006-12-29 | 2008-07-10 | Applera Corporation | Systems and methods for detecting nucleic acids |
| DE102008040513A1 (en) * | 2008-07-17 | 2010-01-21 | BAM Bundesanstalt für Materialforschung und -prüfung | Use of long-wavelength emitting cyanine compound as NIR fluorescence standard and kit for calibration of photoluminescence measurement systems |
| DE102008040513B4 (en) * | 2008-07-17 | 2010-08-26 | BAM Bundesanstalt für Materialforschung und -prüfung | Use of long-wavelength emitting cyanine compound as NIR fluorescence standard and kit for calibration of photoluminescence measurement systems |
| EP2180066A1 (en) * | 2008-10-23 | 2010-04-28 | Honeywell International | Single nucleotide polymorphism genotyping detection via the real-time invader assay microarray platform |
| US8017327B2 (en) | 2008-10-23 | 2011-09-13 | Honeywell International Inc. | Single nucleotide polymorphism genotyping detection via the real-time invader assay microarray platform |
| CN104093857A (en) * | 2011-11-17 | 2014-10-08 | Nvs技术股份有限公司 | Quantitative, highly multiplexed detection of nucleic acids |
Also Published As
| Publication number | Publication date |
|---|---|
| AU2003300271A1 (en) | 2005-08-03 |
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