CN1753993A - Detection of small nucleic acids - Google Patents

Abstract

The present invention relates to compositions and methods for the detection and characterization of interfering RNAs such as micro RNAs (miRNAs) and small interfering RNAs (siRNAs) and other short nucleic acid molecules. More particularly, the present invention relates to improved methods for the detection and quantitation of interfering RNA expression. The present invention further provides for the detection of variants and types of miRNAs and siRNAs.

Description

The detection of small nucleic acids

The application requires the right of priority of U.S. Provisional Application sequence number 60/434,518 (December 18 2002 applying date) and U. S. application sequence number 60/443,814 (January 30 2003 applying date).

Invention field

The present invention relates to RNA interfering (for example Microrna (miRNA) and siRNA (siRNA)) and the composition of other short nucleic acid molecule and the method for detection and sign thereof.More especially, the present invention relates to the detection of RNA interfering expression and the improved method of quantitative analysis.The present invention further provides the varient of miRNA and siRNA and the detection of type.

Background of invention

Microrna (miRNA) is a kind of non-coding RNA of novel type, and they are encoded as short inverted repeats (Ambros, (2001) cell (Cell) 107,823-826 in invertebrates and the vertebrates genome; The contemporary biology of Moss (2002) (Curr Biol) 12, R138-R140).MiRNA is the regulon of said target mrna translation and stability, and is to be identified although most said target mrna has.MiRNA by be incorporated into 3 ' end untranslated zone (UTRs) in antisense paratope point sequence control the translation of said target mrna specifically (Ambros be as preceding; Moss is as preceding; People such as Lagos-Quintana, (2001) science (Science) 294,853-858; People such as Lau, (2001) science (Science) 294,858-862; People such as Lee, (2001) science (Science) 294,862-864).

Some miRNA, for example let-7RNA, miR-1, miR-34, miR-60 and miR-87, between invertebrates and vertebrates, be high conservative, hint and they can discern the multiple site of inferring conservative function and/or multiple target (people such as Lagos-Quintana is as preceding; People such as Lau are as preceding; People such as Lee are as preceding; People such as Pasquinelli, (2000) nature (Nature) 408:86).Little temporary transient RNA (stRNA) lin-4 and let-7 have represented the hypotype (subclass) of the miRNA that identifies out by the genetic analysis of magnificent new rhabditis axei (Caenorhabditis elegans), they be grown adjusting and they oneself control development program, for example nerve connect again (neuronal rewiring) selection of time, the formation of Daucer larva, vaginal orifice forms and the differentiation of end eventually of subcutaneous cell.

MiRNA generally shears from 60 or 70 Nucleotide (foldback) RNA front body structure of turning back, and they are (people such as Grishok, (2001) cell (Cell) 106,23-34 when the expression of miRNA precursor begins sometimes; People such as Hutvagner (2001) science (Science) 93,834-838; People such as Ketting, (2001) gene development (Genes Dev) 15,2654-2659) or in the expression process of profuse miRNA (people such as Lagos-Quintana is as preceding; People such as Lau are as preceding; People such as Lee are as preceding) be detected.Usually, have only a chain of hair clip precursor molecule to be sheared and accumulate, infer that the chances are and avoid RNA and degrade because it is protected by related protein.The protein of these suppositions can mediate the inhibition of translation.MiRNA precursor processing reaction needs Dicer RNAsein and Argonaute family member, and (people such as Grishok is as preceding; People such as Hutvagner are as preceding; People such as Ketting are as preceding).

Except their influences to genetic expression, these are the little RNA in 21-22 Nucleotide scope usually, can find purposes in treatment and drug discovery field, for example as the medicine target or as medicament.Thereby in some cases, about what every kind of miRNA existence are important in the understanding cell.In some cases, before further relatively in the histological types or for example the stimulant of chemistry or physical interventions is used and afterwards the level of miRNA is important.Because relevant siRNA and miRNA can exist with very low amount in cell, the not only responsive but also special detection method of expectation.In addition, use, identify that for example homogeneous assay method, composite determination method or those methods of being suitable for highly parallel automated operation and conditional temperature variation are favourable to the method that is suitable for high flux screening for some.

Although miRNA plays a very important role, lack the detection of miRNA expression and the effective technology of quantitative analysis in the adjusting of genetic expression always.So far, be used for the main method of miRNA quantitative analysis based on gel electrophoresis.Described miRNA detects by Northern trace or the existence by the anti-RNase binary of radioactivity (duplexes).Northern trace and chip hybridization method have relative low assay sensitivity people 2003 such as () Krichevsky, so need the RNA of microgram quality to be used for detecting; In addition, little RNA transfers to strainer can cause the reproducible problem of quantitative analysis, and generally can not revise and be used for high-throughput.In addition, the detection method based on the RNase resistance needs highly radioactive probe.Further, just can not provide enough differences between the closely-related analogs of sequence based on the method for probe hybridization.The alternate method comprises the described miRNA of clone, then to described insertion body examination preface.Though this method is suitable for differentiating the difference of single base between the closely-related miRNA kind, its consuming time and consumption power.

As miRNA, siRNA (siRNA) is to disturb (RNAi) (Cullen, B.R., natural immunity (Nature Immunology) via being called RNA, 3:597-599 (2002)) reaction participates in cytophylactic small RNA molecular, for example antiviral rna.One class siRNA produces by the effect of Dicer enzyme and reticent mixture (RISC) protein complex of RNA inductive, a part (the Khvorova of the described RNAi that double-stranded RNA exists in cell in response, A. wait the people, cell (cell) 115:209-216 (2003)).Another kind of siRNA is a synthetic, the short binary that comprises 21-23 the Nucleotide that distinctive dinucleotides external part is arranged usually, directly introduce cell (Elbashir via transfection or from the expression of introducing carrier, S.M. wait the people, Europe molecular biosciences magazine (EMBO J.) 20:6877-6888 of association (2001)) (Paul, C.P. wait the people, Nature Biotechnol (NatureBiotechnology) 20:505-508 (2002), U.S. Patent Application Publication No. 2003/01481519A1, for all purposes herein integral body quote as a reference).As if in some cases, siRNA is clear and definite sequence always, make them on function and composition similar to miRNA (Elbashir, people such as S.M. are as preceding).Needed is to detect and quantitative analysis miRNA and the effective and accurate method of siRNA level.

Summary of the invention

The present invention relates to the composition of small nucleic acids (for example RNA interfering and other short nucleic acid molecule) and the method for detection and sign thereof.More especially, the present invention relates to the detection of RNA interfering expression and the improved method of quantitative analysis.

For example, the invention provides a kind of method, comprising:, at least a nucleic acid (that nucleic acid that for example contains the sequence that is not complementary to RNA interfering) is hybridized in the RNA interfering target, and detect described detection architecture in order to produce a detection architecture.In some embodiments, described RNA interfering target is miRNA.In other embodiment, described RNA interfering target is siRNA.In some embodiments, described siRNAs is double-stranded.

In some embodiments, described detection architecture comprises invasive cutting structure (invasivecleavage structure).For example, in some embodiments, described nucleic acid comprises first and second oligonucleotide that dispose (configured) for the invasive cutting structure that forms associating miRNA.In some embodiments, described nucleic acid comprises first oligonucleotide that disposes for the invasive cutting structure that forms the described miRNA of associating.In some embodiments, described first oligonucleotide comprises 5 ' part and 3 ' part, and wherein said 3 ' part disposes in order to hybridize in described target sequence, and wherein said 5 ' part disposes in order not hybridize in described target sequence.Adopt in the embodiment of second oligonucleotide at some, described second oligonucleotide comprises 5 ' part and 3 ' part, wherein said 5 ' part disposes in order to hybridize in described target sequence, and wherein said 3 ' part disposes in order not hybridize in described target sequence.In some embodiments, described detection step comprises the use of INVADER test.

In some embodiments, described detection architecture comprises the ring-type oligonucleotide of hybridizing in described little RNA to produce annular detection architecture.In some embodiments, described detection step comprises the rolling-circle replication test.

In some embodiments, described detection step comprises the use that detects test, includes but not limited to: order-checking test, polymerase chain reaction test, cross experiment, employing are complementary to cross experiment, microarray test, the test of pearl array, primer extension test, enzyme mispairing cutting test, branch's cross experiment, NASBA test, molecular beacon test, circle probe test, ligase chain reaction (LCR) test, the test of invasive cutting structure, ARMS test and the sandwich hybridization test of the probe of sudden change.In some preferred embodiments, described detection step is carried out in cell lysate.

In some embodiments, method of the present invention comprises detection second nucleic acid target.In some preferred embodiments, described second nucleic acid target is RNA.In some particularly preferred embodiments, described second nucleic acid target is U6 RNA or GADPH mRNA.

In some embodiments, the nucleic acid that is used to form detection architecture comprises the template with the one or more sites that fully are complementary to little RNA, so that allow described RNA to hybridize in described template and be extended in extension.In some embodiments, described extension is the polymerase chain reaction, and wherein one or more RNA are used as the primer in the polymerase chain reaction.In some such embodiments, for the primer of polymerase chain reaction is provided, the RNA of single type is incorporated into two positions on the template.In other embodiment, two or more RNA are used as primer.In such embodiments, exist (being miRNA composite testing (miRNA multiplex assay)) of the two or more RNA described in the sample represented in the detection of amplified production.Similar methods can be used to ligase chain reaction (LCR), and miRNA described here is used as the oligonucleotide of connection.

In some embodiments, described method comprises the detection of multiple miRNA.In some such embodiments, multiple miRNA comprises the polymorphism of identical miRNA.In other embodiment, multiple miRNA comprises different miRNA (for example Let-7, miR-1, miR-1d, miR-135, miR-15, miR-16, miR-124a or miR125b).

The present invention also provides and has been used for implementing any one test kit of top method.For example, in some embodiments, the invention provides the test kit that comprises the nucleic acid that disposes in order when hybridizing, to form detection architecture in the RNA target sequence.In some embodiments, described test kit disposes in order to detect miRNA.In some preferred embodiments, described test kit disposes for the miRNA that detects Let-7, miR-1, miR-135, miR-15, miR-16, miR1b, miR-124a or miR125b.In some preferred embodiments, described test kit disposes in order to detect the 2nd RNA target and miRNA target altogether.

Description of drawings

Fig. 1 shows in the single reaction and to detect two not synoptic diagram of INVADER oligonucleotide, probe oligonucleotides and FRET (fluorescence resonance energy transfer) (FRET) box of isoallele (for example difference of a Nucleotide).

Fig. 2 demonstration is used for the used exemplary detection structure of some embodiments of the present invention.

Fig. 3 demonstration is used for the second used exemplary detection structure of some embodiments of the present invention.

Fig. 4 demonstration is used for the 3rd used exemplary detection structure of some embodiments of the present invention.

Fig. 5 demonstration is used for exemplary oligonucleotide of the present invention.The base of described target miRNA is underlined small letter.DNA residue in probe or the INVADER oligonucleotide is a normal font.Small letters pointed out 2 '-residue of oxygen-methyl.

Fig. 6 shows the temperature optimization result of experiment of let-7.

Fig. 7 shows the temperature optimization result of experiment of let-7.

Fig. 8 shows detectability (LOD) result of experiment of let-7.

Fig. 9 shows the cross reactivity result of experiment of using let-7 miRNA.

Figure 10 shows miR-1 LOD result of experiment.

Figure 11 shows CLEAVASE enzyme IX and the XII result relatively who uses let-7 miRNA.

Figure 12 shows the partial sequence comparison from the U6 RNA sequence of various microorganisms.

Figure 13 shows the temperature optimization result of experiment of mir-135.

Figure 14 shows mir-135 LOD result of experiment.

Figure 15 contains the diagrammatic representation of the average counter that the detection of let-7 obtains in the cell lysate.

Figure 16 show through or the cell lysate handled through RNAse A in the result of miRNA and mRNA.

Figure 17 shows the result for the invasive cutting test of the effect of the capture oligonucleotide of brachymemma relatively comprised total length.

Figure 18 shows the temperature optimization result of experiment of using the test design of describing among Figure 16.

Figure 19 has shown the temperature optimization result of experiment of using 10 aggressiveness probes and the design of 12 aggressiveness INVADER oligonucleotide.

Figure 20 shows the LOD result of experiment that has compared two substituting oligonucleotide designs.

Figure 21 show compared with some or all of 2 in probe and the INVADER oligonucleotide '-oxygen-methyl residue is substituted by 2 '-result of the effect of deoxidation residue.

Figure 22 shows the result of the invasive cutting test that detects miR-124a.

Figure 23 shows the result of experiment that detects separation five kinds of different miRNA kinds in total RNA of 20 histological types.

Figure 24 shows the experimental result of the effect that probe that test changes and oligonucleotide length detect miRNA.

Figure 25 shows for the design of siRNA detection example invasive cutting oligonucleotide.The small letter residue represents 2 '-the O-methyl.

Definition

In order to make things convenient for the understanding of the present invention, many terms and phrase are defined as follows:

As using ground herein, term " miRNA " refers to Microrna. As used herein, term " miRNA target sequence " refers to miRNA to be detected (for example, in the presence of other nucleic acid). In some embodiments, the miRNA target sequence is the variant of miRNA.

As using ground herein, term " RNA detection architecture " refers to by nucleic acid (for example, oligonucleotides) hybridizes the target in RNA, for example miRNA or siRNA, and the structure that forms. In some embodiments, described nucleic acid is mononucleotide (for example, a zonule (or some zones) being arranged with the larger nucleic acid that comes from described miRNA). In other embodiment, described nucleic acid comprises two kinds of nucleic acid (for example, hybridizing in described miRNA to form the nucleic acid of hair clip (for example, single or two hair clips) structure). In preferred embodiments, the miRNA detection architecture is to use known nucleic acid detection method to detect, and includes but not limited to those structures disclosed herein.

In some embodiments, the RNA detection architecture is further modified after hybridization step. For example, in some embodiments, hybridizing in the described nucleic acid of described RNA provides the template of extending described RNA for by nucleic acid polymerase. Described RNA is hybridized in described nucleic acid and is then used described polymerase to extend. In other embodiment, described nucleic acid plays extra nucleic acid and described RNA hybridization and the template action that is connected.

Term " siRNA " refers to short interfering rna. In some embodiments, siRNA comprises a binary or double-stranded region, and about 18 to 25 nucleotides of each chain of double-stranded region described here are long; Described double-stranded region can be as short as 16 and grow to 29 base-pairs, and length described here is determined by described antisense strand. Usually siRNA contains about two nucleotides to four non-matchings at 3 ' end of each chain. As if siRNA is non-vertebrate and play initiation RNA disturbed and rose the degraded of initiation sequence-specific RNA in the PTGS process in plant crucial intermediate in vertebrate. At least the binary of siRNA or double-stranded region chain is basically with coming from or basically be complementary to target RNA molecule. The chain that is complementary to target RNA molecule is " antisense " chain; Be " justice is arranged " chain and also be complementary to described siRNA antisense strand with the chain that comes from target RNA molecule. A chain of described double stranded region needs not be the precise length of relative chain, thereby a chain can have than relative chain and lacks at least 1 nucleotides, forms " bubble " or relative at least one unpaired base of chain in the relative chain. A chain of described double-stranded region does not need accurately to be complementary to described relative chain; Thereby described chain (preferably described sense strand) can have at least one base mismatch pair.

SiRNA also contains extra sequence; The limiting examples of such sequence comprises catenation sequence or ring, and they couple together two chains in described binary zone. This form of siRNA can refer to " si sample RNA ", " bob folder siRNA ", (weak point described here refers to the binary zone of described siRNA) or " hair clip siRNA ". The other limiting examples of the additional sequences that exists among the siRNA comprises stem and other foldable structure. Described additional sequences can have maybe and can not have known function; The limiting examples of such function comprises the stability that the siRNA molecule increases, and cell purpose (destination) signal perhaps is provided.

As using ground herein, term " research object " and " patient " refer to any organism, comprise plant, microorganism and animal (for example mammal for example dog, cat, domestic animal and people).

As using ground herein, term " INVADER test reagent " or " invasive cutting test reagent " refer to the one or more reagent that detect target sequence, and described regent pack is contained in target sequence and has the lower oligonucleotides that can form the invasive cutting structure. In some embodiments, the described INVADER test reagent reagent that exists of inclusion test invasive cutting structure (for example cutting reagent) further. In some embodiments, described oligonucleotides comprises the first and second oligonucleotides, described the first oligonucleotides comprises the 5 ' part that is complementary to the target nucleic acid first area, and described 5 ' part is complementary to the second area of the target nucleic acid that is positioned at first downstream and contiguous first. In some embodiments, the described 3 ' part of the second oligonucleotides comprises the 3 ' terminal nucleotide that is not complementary to target nucleic acid. In preferred embodiments, 3 of described the second oligonucleotides ' part is comprised of the mononucleotide that is not complementary to target nucleic acid. In some embodiments, the mutual covalent coupling of the first and second oligonucleotides (for example passing through connexon).

In some embodiments, described INVADER test reagent further comprises solid support. For example, in some embodiments, one or more oligonucleotides of described test reagent (for example the first and/or second oligonucleotides is bridge joint or non-bridge joint) are attached to described solid support. In some embodiments, described INVADER test reagent further comprises cushioning liquid. In some preferred embodiments, described cushioning liquid comprises the source (Mn for example of bivalent cation²⁺And/or Mg²⁺Ion). The common separate constituent (for example oligonucleotides, enzyme, buffer solution, target nucleic acid) that consists of the INVADER test reagent is called as " INVADER test reagent component ".

In some embodiments, described INVADER test reagent further comprises the 3rd oligonucleotide, and it is complementary to the target nucleic acid third part of the first target nucleic acid first part upstream.Yet in other embodiment, described INVADER test reagent further comprises target nucleic acid.In some embodiments, described INVADER test reagent further comprises second target nucleic acid.Yet in other embodiment, described INVADER test reagent further comprises the 3rd oligonucleotide, and it comprises the 5 ' part that is complementary to the second target nucleic acid first area.In some specific embodiments, 3 ' part covalency of the 3rd oligonucleotide is connected in second target nucleic acid.In other specific embodiments, second target nucleic acid further comprises 5 ' part, and wherein the described 5 ' part of second target nucleic acid is the 3rd oligonucleotide.In other embodiment, described INVADER test reagent further comprises ARRESTOR molecule (for example ARRESTOR oligonucleotide).

As people such as Eis, natural biology engineering (Nature Biotechnology), ground is described among the 19:673-676 (2001), fully base pairing in the target-specific zone of each probe and portion paired the comprising of the 2 ' oxygen-methylated ARRESTOR oligonucleotide in its 5 ' flap zone, isolated uncut probe and stoped the formation (integral body is quoted as a reference) of X-structure in the secondary reaction herein.

In some preferred embodiments, described INVADER test reagent further comprises the reagent that detects the nucleic acid cleaved products.In some embodiments, the one or more oligonucleotide in the described INVADER test reagent comprise marker.In some preferred embodiments, described first oligonucleotide comprises marker.In other embodiment preferred, described the 3rd oligonucleotide comprises marker.In particularly preferred embodiments, described reagent comprises the first and/or the 3rd oligonucleotide with the group mark that produces FRET (fluorescence resonance energy transfer) (FRET) effect.

In some embodiments, one or more described INVADER test reagents provide with form (that is, being used in preceding (premeasured) of mensuration of a step in the program that does not redeterminate or the distribute again) form of (predispensed) before distributing.In some embodiments, the INVADER test reagent component of selection be " blended " and " distribution before " together.In preferred embodiments, the test reagent component before the distribution is before the distribution, and is provided in the reaction vessel (include but not limited to reaction tube or for example the hole in the microtitration flat board).In particularly preferred embodiments, the INVADER test reagent component before the distribution is (for example, dehydration or cryodesiccated) of drying in reaction vessel.

In some embodiments, described INVADER test reagent can be provided as test kit.As use herein, term " test kit " refers to any delivery system of delivered substance.In the context of reaction test, such delivery system (for example comprises the permission reaction reagent, oligonucleotide in the appropriate containers, enzyme etc.) and/or support substance is (for example, damping fluid is carried out printed instructions of test or the like) storage from the three unities to another place, the system that transports or send.For example, described test kit comprises one or more encapsulation object (for example, box) that contain correlated response reagent and/or support substance.As use herein, term " (fragmented) test kit that separates " refers to the delivery system that comprises two or more containers that separate, and wherein each container contains an inferior part of whole reagent constituents.Described container is delivered to the susceptor of expection together or respectively.For example, first container can contain the enzyme that is useful on test, and second container contains oligonucleotide.Term " test kit that separates " is intended to include but not limited to contain the test kit of analyte specific reagent under federal food, medicine and the regulation and control of makeup bill the 520th chapters and sections (ASR ' s).Certainly, any delivery system that comprises two or more containers that separate all is included among the term " test kit that separates ", and wherein each described container contains an inferior part of whole reagent constituents.On the contrary, " test kit of associating " refers to the delivery system (the single box that for example, has held each desired components) that contains all components of a reaction test in single container.Term " test kit " comprises separately and test kit associating.

In some embodiments, the invention provides the INVADER test kit of the component that comprises one or more enforcement the present invention needs.For example, the invention provides to be used to store or send and carry out the test kit that INVADER tests required enzyme and/or reactive component.Described test kit can comprise any and whole component that test is required or expect, include but not limited to, described reagent itself, damping fluid, contrast agents are (for example, tissue sample, the positive and negative control target oligonucleotide or the like), solid-state upholder, marker, written and/or illustrate book and product information, inhibitor, mark and/or detection reagent, pack environment control thing (for example, ice, siccative or the like) or the like.In some embodiments, described test kit provides an inferior cover essential component, expects that wherein the user will provide remaining component.In some embodiments, described test kit comprises two or more independently containers, and wherein each container has been collected the described component of an inferior cover to be sent.For example, first container (for example, box) can contain enzyme (for example, structure specificity nickase in suitable store buffer liquid and the container), and second box can contain oligonucleotide (for example, INVADER oligonucleotide, probe oligonucleotides, contrast target oligonucleotide or the like).

Refer to as the term " marker " that uses herein and can be used to provide the effect that can detect (preferably quantitative) and can be attached to nucleic acid or proteinic any atom or molecule.Described marker includes but not limited to dyestuff; The radio-labeled thing (for example ³²P); Conjugated group (biological example element); Haptens is digoxgenin for example; Luminous, phosphorescent or fluorescigenic group; Quality tab and fluorescence dye are united separately or with the group that can pass through FRET (fluorescence resonance energy transfer) (FRET) inhibition or mobile emission spectrum.Described marker can provide by fluorescence, radioactivity, colorimetry, weight determination, X-ray diffraction or absorption, magnetic force, enzymic activity, be subjected to the quality of quality influence or the feature of behavior (for example, MALDI time-of-fight mass spectrometry; Fluorescence polarization) or the like detectable signal.Marker can be that charged group (plus or minus electric charge) maybe can be electroneutral.Described marker can comprise nucleic acid or protein sequence or be made up of it, be detectable as long as comprise the sequence of described marker.

As using ground herein, the term " distinct " that relates to signal refers to the signal that can distinguish mutually, for example pass through mass spectrum property for example fluorescent emission wavelength, color, absorbancy, quality, size, fluorescence polarization characteristic, electric charge or the like, perhaps by with the interactional ability of another group, for example with chemical reagent, enzyme, antibody or the like.

As using ground herein, term " complementary " or " complementation " are used in reference to because of the relevant polynucleotide of base pairing rules (that is, for example the nucleotide sequence of oligonucleotide or target nucleic acid).For example, be complementary to sequence " 3 '-T-C-A-5 ' " for sequence " 5 '-A-G-T-3 ' ".Complementation can be " part ", and wherein having only the base of some nucleic acid is paired according to base pairing rules.Perhaps, " completely " or " whole " complementation can be arranged between the nucleic acid.The complementary degree has significant effect to the efficient and the intensity of hybridizing between the nucleic acid chains between the nucleic acid chains.This is at amplified reaction and depend between the nucleic acid particularly important in the bonded detection method.Any term also can be used for relating to independent Nucleotide, especially in the context of polynucleotide.For example, contrast with the described oligonucleotide of remainder and the described complementation between the described nucleic acid chains or compare, special Nucleotide is complementary to because of it or lacks the Nucleotide that is complementary in another nucleic acid chains and noted in the oligonucleotide.

Term " homology " refers to identical degree with " homologous ".Portion homologous or complete homology can be arranged.Homeologous sequence be a sequence with the another one sequence be less than 100% identical.

As using ground herein, term " hybridization " is used in reference to the pairing of complementary nucleic acid.The intensity (that is the intensity of uniting between the described nucleic acid) of hybridization and hybridization is subjected to complementary degree between for example described nucleic acid, about the influence of the factors such as Tm of the heterozygote of the severity of condition and formation." hybridization " method comprises the annealing of the nucleic acid of a nucleic acid and another complementary nucleic acid (promptly having complementary nucleotide sequence).Two polymers that contain the nucleic acid of complementary sequence are sought mutually and are the extensive phenomenons of checking by base pairing interaction annealed ability.Marmur and Lane, people such as Proc.Natl.Acad.Sci.USA 46:453 (1960) and Doty, after the original observed of Proc.Natl.Acad.Sci.USA 46:461 (1960) to " hybridization " process, this method is refined into the basic tool of modern biology.

Complement as the nucleotide sequence that uses herein refers to a kind of oligonucleotide, and when making 5 of a sequence ' end and 3 of another sequence ' terminal the pairing with the nucleotide sequence comparison, described oligonucleotide is in " antiparallel combination ".Usually some base that can not find in the natural acid can be included among the nucleic acid of the present invention, comprises for example xanthoglobulin and 7-denitrification guanine (deazaguanine).Complementation needs not be perfectly (perfect); Stable binary can contain base pair that mismatches or the base of not mating.Technician in the nucleic acid technical field considers rule of thumb to comprise that following many variablees just can determine disome stability, for example the sequence of the length of oligonucleotide, based composition and oligonucleotide, ionic strength and mismatch the incidence of base pair.

As using ground herein, term " Tm " is used in reference to " temperature of fusion ".Described temperature of fusion is the temperature that a group double-strandednucleic acid half is dissociated into strand.Some equations that calculate nucleic acid Tm are well known in the art.Point out ground as the canonical reference document, when nucleic acid is in the 1M NaCl aqueous solution, (for example see, Anderson and Young, quantitative filtration hybridization (Quantitative Filter Hybridization in the nucleic acid hybridization, in Nucleic Acid Hybridization) (1985)), the simple estimation of Tm value can be passed through equation: Tm=81.5+0.41 (%G+C) and calculate.Other reference (for example Allawi and SantaLucia, biological chemistry (Biochemistry) 36:10581-94 (1997)) comprises complicated more calculating, and it has considered structure and environment and sequence signature for the calculating of Tm.

Term " gene " refers to the essential control of the generation of the RNA (for example, rrna or transfer RNA), polypeptide or the precursor that include non-encoding function and the dna sequence dna of encoding sequence.Described RNA or polypeptide can be encoded by complete encoding sequence or by any part of described encoding sequence, as long as the activity or the function of expectation are saved.

The gene or the gene product of the gene that term " wild-type " has when referring to and separating from the source of natural generation or the feature of gene product.Wild type gene is to observe in a colony the most continually, and thereby subjectively is appointed as " normally " or " wild-type " form of gene.On the contrary, term " modification ", " sudden change " or " polymorphism " refer to when comparing with wild type gene or gene product, have shown the gene or the gene product of the modification (i.e. the characteristic of Gai Bianing) of sequence and/or functional performance.Mutant that it should be noted that natural generation can be isolating; These mutant have during by them and wild type gene or gene product comparison change feature the fact and identified.

Refer to the dna sequence dna of the heterologous sequence that contains expectation herein as the term " recombinant DNA carrier " that uses.For example, although described term is not limited to the use of the sequence of the sequence of expression or the expression product of encoding, in some embodiments, described heterologous sequence be a kind of encoding sequence with host organisms in the essential suitable dna sequence dna of expression that duplicates (or the encoding sequence that is connected effectively in the special host organisms) of encoding sequence.Express essential dna sequence dna in the prokaryotic cell prokaryocyte and comprise promotor, randomly operon sequence, ribosome bind site and other possible sequence.Known promotor, polyadenous glycosidation signal and the enhanser of utilizing of eukaryotic cell.

Be defined as comprising the molecule of two or more deoxyribonucleotides or ribonucleotide herein as the term " oligonucleotide " that uses, at least 5 Nucleotide preferably, more preferably about at least 10-15 Nucleotide, more preferably about at least 15 to 30 Nucleotide.Definite big young pathbreaker depends on several factors, and these factors depend on the final function or the use of described oligonucleotide conversely.Described oligonucleotide can produce by any way, comprises chemosynthesis, dna replication dna, reverse transcription, PCR or their associating.

Be attached to the such mode of its adjacent 3 ' oxygen and react because mononucleotide connects via a phosphodiester in a direction with 5 ' phosphate of a mononucleotide pentose ring, to make oligonucleotide, if 5 ' phosphate of oligonucleotide is not connected in 3 ' oxygen of mononucleotide pentose ring, this end just is called as " 5 ' end ", if its 3 ' oxygen is not connected in 5 ' phosphate of mononucleotide pentose ring subsequently, just be called as " 3 ' end ".As using ground herein, even if a bigger oligonucleotide inside, nucleotide sequence also we can say to have 5 ' and 3 ' end.If along nucleic acid chains with 5 ' move to 3 ' direction, 3 of first area ' end is before 5 ' end of second area, it is another regional upstream that the first area of described nucleic acid chains just is said to be.

When of the different zones annealing of two different non-overlapped oligonucleotide with identical linear complementary nucleotide sequence, and when 3 of an oligonucleotide ' end points to another 5 ' terminal, the former can be called as " upstream " oligonucleotide, and the latter is called as " downstream " oligonucleotide.Similarly, when two eclipsed oligonucleotide hybridizations in identical linear complementary nucleotide sequence, settling first oligonucleotide to make that its 5 ' end is 5 of second oligonucleotide ' terminal upstream, when 3 ' end of first oligonucleotide is the upstream of second oligonucleotide 3 ' end, first oligonucleotide can be called as " upstream " oligonucleotide, and second Nucleotide can be called as " downstream " oligonucleotide.

Term " primer " refers to the following time of condition that can make primer extension initial when placing, and can play the oligonucleotide of synthetic starting point.Oligonucleotide " primer " can produce naturally when time in the restrictive diges-tion at purifying, perhaps can produce synthetically.

Select primer to be complementary to the chain of described template specificity sequence with " basically ".For the prolongation of primer takes place, primer must be fully complementary to hybridize in template strand.Primer sequence does not need to reflect the definite sequence of described template.For example, non-complementary nucleotide fragments can be attached to 5 ' end of described primer, and the remainder of primer sequence is complementary to described chain basically.Incomplementarity base or longer sequence can be dispersed within the described primer as long as primer sequence has with the complementation fully of the template sequence that will hybridize and therefore forms the templa-primer mixture with the synthetic primer that extends.

As the term " cutting structure " that uses herein, refer to structure by the interaction formation of at least one probe oligonucleotides and target nucleic acid, form the structure that comprises binary, resulting structures can be cut by the cutting mode that includes but not limited to enzyme.Compare with the nucleic acid molecule that is the substrate of the non-specific cutting of reagent by for example phosphodiesterase, it is not considered secondary structure and cuts nucleic acid molecule (promptly, do not need the formation of couple structure), described cutting structure is the substrate by described cutting mode specificity cutting.

As the term " cutting mode " that herein uses or " cutting reagent " refer to can cut as described in any method of cutting structure, include but not limited to enzyme." structure specific nucleic acid enzyme " or " structure specific enzymes " are specific secondary structures and cut the enzyme of these structures in the identification nucleic acid molecule.Cutting mode of the present invention responds the formation of cutting structure and cuts nucleic acid molecule; It is dispensable that described cutting method any privileged site in described cutting structure cuts described cutting structure.

Described cutting mode can comprise the nuclease that multiple source provides, comprise the CLEAVASE enzyme (the 3rd ripple science and technology, Madison, WI), FEN-1 endonuclease (comprising RAD2 and XPG protein), Taq archaeal dna polymerase and e. coli dna polymerase I.Described cutting mode can comprise the enzyme (for example Taq archaeal dna polymerase (DNAP), e. coli dna polymerase I) with 5 ' nuclease.Described cutting method also can comprise the archaeal dna polymerase that has 5 ' nuclease but lack the modification of composite reactive.Be fit to use the embodiment of the cutting mode that in the inventive method and test kit, uses at U.S. Patent number 5,614,402,5,795,763,5,843,669, provide among PCT application number WO 98/23774, WO02/070755A2 and the WO0190337A2, they each integral body is herein quoted as a reference.

When the enzyme that relates to 5 ' nuclease for example and when being used, term " heat-staple " refers to that described enzyme is functional or activated at elevated temperatures, promptly, under about 55 ℃ or higher temperature (for example, including but not limited to 60 ℃, 65 ℃, 70 ℃, 75 ℃, 80 ℃, 85 ℃ or 90 ℃).

As the term " cleaved products " that uses herein, the product that the cutting method that refers to and the reaction of cutting structure (that is, with the processing of cutting method to cutting structure) produce.

Term " non-target cleaved products " refers to the product of the cleavage reaction that does not derive from described target nucleic acid.As top discussion ground, in certain methods of the present invention, the cutting of described cutting structure generally occurs in the probe oligonucleotides.The fragment of cutting the probe oligonucleotides of generation by this target nucleic acid dependency is " a non-target cleaved products ".

Term " probe oligonucleotides " refers in the context of INVADER test reaction: interact with target nucleic acid and having or do not have the INVADER oligonucleotide in the presence of form the oligonucleotide of cutting structure.When annealing with target nucleic acid, described probe oligonucleotides and target form cutting structure, and cutting occurs within the described probe oligonucleotides.

Term " INVADER oligonucleotide " refers to the oligonucleotide of hybridizing in target nucleic acid near the position the hybridization zone between probe and the target nucleic acid, wherein said INVADER oligonucleotide comprise and described probe and target between hybridization zone eclipsed part (for example, chemical group or Nucleotide---no matter be complementary to or be not complementary to that target).In some embodiments, described INVADER oligonucleotide contains basically and the identical sequence of sequence that is positioned at probe oligonucleotides 5 ' end at its 3 ' end.

Term " ARRESTOR molecule " refers in order to stop one or more reactive components to participate in subsequently effect or reaction and join or be included in the reagent of invasive cleavage reaction.This can be undertaken by isolated or some reactive components of inactivation (for example, by combining or base pairing with nucleic acid component, perhaps by combining with protein component).Term " ARRESTOR oligonucleotide " (be also referred to as and capture oligonucleotide) refers to one or more aspects (for example, first reaction and/or any subsequent reaction or the effect in order to stop or catch any reaction; This is not intended to described ARRESTOR oligonucleotide is limited to any special reaction or reactions steps) and be included in oligonucleotide in the invasive cleavage reaction.This can be undertaken by more isolated reactive components (for example, combining with another nucleic acid base pairing or with protein ingredient).Yet this is not intended to described term so is confined to just make the segregate situation of reactive component.

Refer in the INVADER test to the cutting of response probe oligonucleotides as the term " box (cassette) " that uses herein and to produce the oligonucleotide that detectable signal disposes or the associating of oligonucleotide.In preferred embodiments, described box is hybridized the non-target cleaved products that obtains in the cutting from described probe oligonucleotides, to form the second invasive cutting structure, described box can be cut then.

Secondary cleavage reaction in some embodiment preferred of the present invention comprises the use of FRET box.Such molecule provides the cut sequence of secondary target (secondary reaction target (or SRT)) and FRET mark, allows the homogeneous phase of continuous invasive cleavage reaction to detect (that is, not having separation or other operation of product after the described reaction).Other embodiment preferred has been used the secondary reaction system, and wherein said FRET probe and synthetic target are provided as oligonucleotide separately.The 5 ' redundant chain (flap) that is cut from primary reaction plays the invasion oligonucleotide in secondary reaction, wherein they are incorporated into suitable secondary reaction target (SRT).

In some embodiments, described box is the single oligonucleotide that comprises hair clip part (that is, a zone, wherein the box oligonucleotide part is hybridized second part in identical oligonucleotide under reaction conditions, to form binary).In other embodiment, box comprises at least two and includes the oligonucleotide that can form the complementary portion of binary under reaction conditions.In preferred embodiments, described box comprises marker.In particularly preferred embodiments, described box comprises the group of the mark that produces FRET (fluorescence resonance energy transfer) (FRET) effect.

When being used when relating to nucleic acid primer, term " strand basically " means that the double-stranded substrate that exists with the double-strandednucleic acid form that keeps together to interact by base pairing in the chain compares, and described substrate molecule mainly exists with single-chain nucleic acid.

As using ground herein, phrase " oligonucleotide that is not amplified detects test " refers to the detection test of such structure, wherein for detect or do not have be not amplified (for example, passing through PCR) special target sequence is (for example, miRNA, SNP, tumor-necrosis factor glycoproteins or the like) existence, and need not to produce the replisome of described target sequence." nonamplifie oligonucleotide detect test " is passable, and for example, amplification is used to indicate the signal that has or do not have special target sequence or polypeptide existence among the target sequence, as long as described target sequence is not replicated.

As using ground herein, phrase " oligonucleotide of non-amplification detects test " refers to the detection test of such structure, wherein exist, and need not to produce the replisome of described target sequence in order to have detected or not had target sequence (for example miRNA, SNP, tumor-necrosis factor glycoproteins or the like)." oligonucleotide of non-amplification detects test " is passable, and for example, amplification is used for indicating target sequence to have or does not have special target sequence or the signal of polypeptide existence, as long as target sequence does not duplicate.

Refer to the difference of nucleotide sequence between two nucleic acid herein as the term " sequence variations " that uses.For example, the mutant form of wild-type structure gene and this wild-type structure gene can and/or lack owing to the alternative of a base, or the existence of the insertion of one or more Nucleotide, and different on sequence.Two forms of this of described structure gene it is said different on sequence.Second mutant form of described structure gene can exist.This second mutant form it is said all different with first mutant form of described gene with wild type gene on sequence.

Refer to nucleic acid fragment effect by for example 5 ' nuclease release from the bigger nucleic acid fragment of for example oligonucleotide as the term " releases " that uses herein, to such an extent as to the fragment of release no longer is covalently attached on the remaining oligonucleotide.

Refer to the Michaelis-Menten constant of enzyme as the term " Km " that uses herein, be defined as specific enzyme and in enzymatic reaction, produce half the concentration of specific substrates of its maximum rate.

Refer to Nucleotide modification or that non-natural take place as " nucleotide analog " that uses herein, include but not limited to: for example 7-deazapurine (that is 7-denitrogenation-dATP and 7-denitrogenation ,-dGTP) of the interactional analogue of accumulation with change; The base analogue (for example describe in the U.S. Patent number 6,001,983 of Iso-C and Iso-G and S.Benner and herein quote as a reference other off-gauge base pair) that alternate hydrogen bonding configuration is arranged; Non-hydrogen bonding analogue (for example, nonpolar, aromaticity nucleoside analog, for example B.A.Schweitzer and E.T.Kool, organic chemistry magazine (J.Org.Chem.), 1994,59,7238-7242, B.A.Schweitzer and E.T.Kool, Journal of the American Chemical Society (J.Am.Chem.Soc.), 1995,117,2 of 1863-1872 description, the 4-difluoro toluene; They each is quoted as a reference herein); " general " base is 5-base indoles and 3-nitro-pyrrole for example; And general purine and pyrimidine (for example are respectively " K " and " P " Nucleotide; P.Kong waits the people, nucleic acids research (Nucleic Acids Res.), and 1989,17,10373-10383, people such as P.Kong, nucleic acids research (Nucleic Acids Res.), 1992,20,5149-5152).Nucleotide analog is included in the Nucleotide that has modification in the glycosyl group, for example di-deoxynucleoside acid and 2 '-oxygen-methyl nucleotide.Nucleotide analog comprises the modified forms of deoxyribonucleotide and ribonucleotide.

Term " polymorphic locus " is having shown between a group member of existing in the colony locus of variation (for example, prevailing allelotrope have be less than 0.95 frequency).Compare, " monomorphism locus " be between the described group member seldom or do not have a genetic loci that variation is observed (being commonly considered as the locus that prevailing allelotrope in described crowd's the gene pool surpasses 0.95 frequency).

As the term " microorganism " that uses herein to such an extent as to be meant a too little visual inspection of organism less than, include but not limited to bacterium, virus, protozoon, fungi and ciliate.

Term " microbial gene sequence " refers to the gene order that derives from microorganism.

Term " bacterium " refers to any bacterial species that comprises eubacterium and archeobacteria class.

Term " virus " refer to can not self-replicating (that is, duplicating the use that needs host cell mechanism) obligate, supermicroscopic, cytozoon.

Term in this specification sheets and the claim " sample " is with its wide significance and be used.It is intended to comprise sample or culture (for example, microorganisms cultures) on the one hand.On the other hand, it is intended to comprise biological and sample environment.Sample can comprise the sample in synthetic source.

Biological sample can be an animal, comprises the mankind, fluid, solid (for example, ight soil) or tissue, and liquid and solid food and feeds product and the composition for example byproduct and the rubbish of milk preparation, plant, meat and meat.Biological sample can be from the domestic animal of all each sections, and wild beast or wildlife obtain, and includes but not limited to for example animal of ungulate, bear, fish, Lagomorpha, mouse or the like.

Environmental sample comprises surrounding material, for example surface mass, soil, water and production piece, and derive from food and milk preparation machining tool, equipment, instrument, utensil, disposable and non-once project.These examples are not interpreted as restriction and can be applicable to sample type of the present invention.

Term " source of target nucleic acid " refers to any nucleic acid (sample of RNA (for example, miRNA) or DNA) that contains.The particularly preferred source of target nucleic acid is a biological sample, includes but not limited to blood, saliva, cerebrospinal fluid, peritoneal fluid, milk, lymph, phlegm and seminal fluid.

If oligonucleotide exists with the volumetric molar concentration higher than another oligonucleotide (or target nucleic acid sequence), just say that oligonucleotide with respect to another oligonucleotide (or target nucleic acid sequence) and " excessive " exist.When the oligonucleotide of for example probe oligonucleotides existed with respect to the concentration excess of complementary target nucleic acid sequence in cleavage reaction, described reaction can be used to the amount of pointing out that described target nucleic acid exists.Usually, when excessive the existence, described probe oligonucleotides will exist with at least 100 times of molar excess; When described target nucleic acid sequence existed with about 10fmole or amount still less, usually each probe oligonucleotides of 1pmol can be used at least.

The sample of " suspection contains " first and second target nucleic acids can contain any one of these two target nucleic acid molecules, and perhaps two all contain or do not contain.

This sentences term " reactant " its wide significance and is used.Described reactant can comprise, for example, and enzyme reaction thing, chemical reactant or light (for example, the known destruction oligonucleotide chain of UV-light, particularly short UV light).Any reagent that can shorten (for example, cutting) with oligonucleotide reaction or prolong described oligonucleotide all is included among the term " reactant ".

As using ground herein, term " purifying " or " wanting purifying " refer to the removal of pollutent from sample.For example, in some embodiments, recombinant C LEAVASE enzyme nucleic acid expression is in bacterial host cell, and described nuclease carries out purifying by the removal of host cell proteins matter; Therefore the per-cent of these recombinant nucleic acid enzymes increase in sample.

Refer to that proteinic fragment as the term " part " (for example in " given proteinic part ") when relating to protein that uses herein.Described clip size scope can deduct from 4 amino-acid residues to complete aminoacid sequence an amino acid (for example, 4,5,6 ..., n-1).

Refer to oligonucleotide, Nucleotide or polynucleotide and their fragment or part as the term " nucleotide sequence " that uses herein, and the DNA or the RNA in genome or synthetic source, they can be single or double-stranded, and representative has justice or antisense strand.Similarly, refer to peptide or protein sequence as " aminoacid sequence " that uses herein.

As using ground herein, term " purifying " or " purifying basically " refer to the molecule of nucleic acid (or amino acid) sequence, described molecule from their natural surroundings be moved out of, separated or be separated, and be at least 60% free, preferably 75% free and most preferably 90% be free on and they natural other relevant components." isolating polynucleotide " or " isolating oligonucleotide " are so be purified polynucleotides basically.

Mean as the term " continuous chain of nucleic acid " that uses herein have successive, covalently bound skeleton structure and do not have the chain of the nucleic acid of breach or other interruption.The tendency of each nucleotide base part, no matter base pairing, strand or mismatch, not the key element of the definition of continuous chain.The skeleton of described continuous chain is not limited to ribose-phosphoric acid ester or the ribodesose-phosphate ester components that finds in the nucleic acid of unmodified of natural generation.Nucleic acid of the present invention can comprise the modification of described skeleton structure, includes but not limited to the ribose residue (for example, 2 '-oxygen-methylribose) of thiophosphatephosphorothioate residue, phosphonic acid ester residue, 2 ' replacement and the residue that contains optional sugar (for example, pectinose).

Refer to a zone of double-strandednucleic acid herein as the term " successive binary " that uses, wherein in described binary, do not interrupt (that is, the described base pair along described binary is not twisted to hold a gap, protrude or mismatch) in the traveling process of base pair in the constraint in continuous binary zone.As described in only referring to as term as described in using herein in the binary as described in the arrangement of base pair, and do not have the successional hint of described nucleic acid chains skeleton part.The base pairing that do not interrupt is arranged but binary nucleic acid jaggy is within the definition of binary continuously in one or two chains.

Term " binary " refers to the state of nucleic acid, and wherein the base portion of Nucleotide is incorporated into their complementary base that is arranged on second chain on chain by hydrogen bonding.The condition that is in the binary form has reflected the state of the described base of nucleic acid.Rely on base pairing, described nucleic acid chains has generally also been supposed double-helical tertiary structure, has big and ditch.The hypothesis of described spiral form hints in the behavior that becomes binary.

Term " template " refers to a chain of nucleic acid, and the complementary replisome is constructed by ribonucleoside triphosphote by the activity of the dependent nucleic acid polymerase of template on described chain." end " chain is described and be described as to described template strand within the binary according to convention.Similarly, " top " chain is often described and be described as to described non-template chain.

Detailed Description Of The Invention

The present invention relates to the short nucleic acid molecule of Microrna (miRNA) or other, for example, siRNA, composition and the method for detection and signature analysis.The invention provides the improved method that detection, signature analysis and quantitative analysis miRNA express.In some embodiments, the invention provides and comprise nucleic acid is joined the method that miRNA expresses with the detection miRNA that help to detect.Gained " miRNA detection architecture " uses any suitable method to detect then, includes but not limited to those methods disclosed herein.Though following description concentrates on detection and the quantitative analysis of miRNA, be to be understood that the present invention has also found the purposes with other short nucleic acid molecule (for example, length is less than for example DNA and the RNA of 50,40,30 or 20 Nucleotide).

1.miRNA the formation of detection architecture

In some embodiments, the invention provides the method for miRNA detection architecture that produce to help miRNA to detect.MiRNA is very little (about 21 Nucleotide), thereby to use standardized hybridizing method to detect be difficult.In some embodiments, method of the present invention comprises nucleic acid molecule is joined miRNA (for example, via hybridization, extend or connect) to produce detection architecture.Such detection architecture can use any suitable method to detect then.

In a special embodiment, for the detection of miRNA has produced the detection architecture of describing among Fig. 2.In this embodiment, two oligonucleotide and described miRNA annealing is to form a dicyclo or " dumbbell " spline structure.Described dumbbell structure is by the terminal double-strandednucleic acid in a bigger zone that produced of the described miRNA that is extended with the oligonucleotide double-stranded region.In some embodiments, the Nucleotide between each end of described miRNA has been extended 2 to 5.In some embodiments, the end of described oligonucleotide comprises the extra nucleotide sequence of not hybridizing in described miRNA.In some embodiments, these extra sequences have formed invasive cutting structure (for example, INVADER test invasive cutting structure).In some embodiments, the invasive cutting structure detects (for example seeing following description) by the INVADER test.

In other embodiment, for the detection of miRNA has produced the detection architecture of describing among Fig. 3.In this embodiment, oligonucleotide and described miRNA annealing is to produce bow-shaped structural.Described miRNA gathers together the end of oligonucleotide, brings than there not being described miRNA to have bigger efficient.In some embodiments, the end of described oligonucleotide has comprised to extend to and exceeds miRNA and do not hybridize extra sequence in described mRNA.In some embodiments, these extra sequences have formed invasive cutting structure (for example, INVADER test invasive cutting structure).In some embodiments, the invasive cutting structure detects (for example seeing following description) by described INVADER test.In other embodiment, after the cutting of INVADER test invasive cutting structure, the end that obtains is connected to form ring structure.In other embodiment, oligonucleotide hybridization is in miRNA, makes that the end of described oligonucleotide is brought to nearby (for example, hybridizing the contiguous Nucleotide in described miRNA) and is connected then.

In embodiment further, produced the detection architecture of describing in Figure 16 and 17.In this embodiment, probe or INVADER oligonucleotide are extended to produce single hairpin loop or " half dumbbell " structure.In some embodiments, the end of described oligonucleotide comprises the extra nucleotide sequence of not hybridizing in described miRNA.In some embodiments, these additional sequences form invasive cutting structure (for example, INVADER test invasive cutting structure).In some embodiments, the invasive cutting structure detects (for example seeing following description) by described INVADER test.

In other embodiment, these extra sequences are complementary to and join reaction mixture to stablize the extra oligonucleotide of cutting structure, for example INVADER test invasive cutting structure (Fig. 4).

In some embodiments, use rolling-circle replication test (for example seeing the following description of rolling-circle replication) to detect as the ring structure of above-mentioned generation.

In embodiment further, detection architecture is from having the long oligonucleotide (for example, greater than 50,100,1000 or more a plurality of Nucleotide) of the regional homology of one or more weak points to produce with miRNA.One or more miRNA are hybridized in described oligonucleotide to produce described detection architecture.In some embodiments, the extension of these detection architecture by miRNA (for example, via connecting or polyreaction RT-PCR for example) detects.In some embodiments, these detection architecture detect further by the hybridization with the oligonucleotide that is coupled to (for example microballoon or other surface or structure) solid support.

In some embodiments, the oligonucleotide that is used to form detection architecture comprises one or more nucleotide analogs.For example, in some embodiments, 2 '-oxygen-methyl nucleotide is utilized.The present invention is not limited to special mechanism.Certainly, optional to the understanding of described mechanism in implementation the present invention.Yet, can expect 2 '-oxygen-existence of methyl base increased the stability of the detection architecture of described hybridization, and helped further detection method.

2. the detection of RNA interfering

In some embodiments, the invention provides the method that detects miRNA.The present invention is not limited to specific detection test.Any suitable method can be utilized, and includes but not limited to those methods disclosed herein.

In some embodiment preferred of the present invention, the miRNA detection method is quantitative.The present invention is not limited to special mechanism.Certainly, optional to the understanding of described mechanism in implementation the present invention.Yet, can expectation body in the level of special miRNA be relevant with gene expression dose from their isoformgenes.The present invention thereby the method that provides genetic expression with specific gene (for example, involved in diseases state or metabolic gene) to be mutually related miRNA.For example, in some embodiments, method of the present invention is used to measure the existence of unusual (for example, high or low) level of specific miRNA, or measures the effect that a kind of intervention (for example, medicine) is expressed miRNA.In other embodiment, (for example, from expression vector, transgenic constructs, transfection or the like) detects to characterize the efficient of miRNA expression system to heterology miRNA.

In some embodiments, the invention provides the method that detects specific miRNA (for example, miRNA such as mir-1 or mir-135).In other embodiment, method of the present invention is used to distinguish the varient (for example, polymorphism or sudden change) among the specific miRNA.In embodiment further, the invention provides lysing cell to detect the method that miRNA exists.

The A.INVADER test

In some embodiments, described INVADER test is used to the detection of miRNA.In some embodiments, described INVADER test comprises formation and depends on the nucleic acid cleavage structure that target nucleic acid exists, and cuts described nucleic acid cleavage structure to discharge unique cleaved products.For example, the activity of 5 ' nuclease is used to cut described target dependency cutting structure, and the cutting of gained cleaved products or described cutting structure is the indication that the specific target nucleotide sequence exists in the sample.When one or two (or more than two) chain of nucleic acid, perhaps oligonucleotide, all hybridize in target nucleic acid chain so that their when forming eclipsed invasive cutting structure, as described below, the invasive cutting can take place.By the interaction of cutting reagent (for example, 5 ' ribozyme) and upstream oligonucleotide, can make described cutting reagent, the mode that is produced with the fragment of uniqueness is at inner site cutting downstream oligonucleotide.Such embodiment is called as INVADER test (the 3rd ripple science and technology) and at U.S. Patent number 5,846, and 717,5,985,557,5,994,069,6,001,567,6,090,543,6,348,314 and 6,458,535, WO 97/27214, WO98/42873, people such as Lyamichev, Nature Biotechnol (Nat.Biotech.), people such as 17:292 (1999), Hall, PNAS, USA, 97:8272 has description in (2000), for they each of all purposes is quoted as a reference herein).

Described INVADER test is by the enzyme cutting of structure specific enzymes to specificity structure, and the hybridization of detection probes and target (is seen INVADER test, the 3rd ripple science and technology; For example see U.S. Patent number 5,846,717,6,090,543,6,001,567,5,985,557,5,994,069,6,090,543,6,348,314,6,458,535, Application No. 20030186238 (sequence number 10/084839), 20030104378A1 (sequence number 09/864636), people such as Lyamichev, natural biology science and technology (Nat.Biotech.), 17:292 (1999), people such as Hall, PNAS, USA, 97:8272 (2000), WO97/27214 and WO98/42873 are for they each of all purposes is quoted as a reference herein).

The mixture that described INVADER test is formed by the hybridization of overlapping oligonucleotide probe by utilization structure specific enzymes (for example FEN endonuclease) cutting, the DNA of detection specificity and RNA sequence (seeing, for example Fig. 1).A temperature that raises and a described probe excessive can make the multiple probe at each target to be cut exist and not have temperature cycle.In some embodiments, the probe of these cuttings instructs the cutting of second label probe then.Described secondary probe oligonucleotides can be with being carried out 5 ' end mark by the fluorescein of dye inside cancellation.Once cutting, the described fluorescein-labeled product of cancellation that goes can use the dull and stereotyped reading apparatus of standard fluorescence to detect.

Described INVADER test has detected amplification and amplification RNA and the specific sequence among the DNA, sudden change and SNP, comprises genomic dna.In the pictorialization embodiment, two concatenation step that all produce and increase described target-specific signal then (primary and secondary reaction) are used in described INVADER test in Fig. 1.For convenience, allelotrope described in the following discussion is described to wild-type (WT) and mutant (MT), even this term and be not suitable for all genovariation.In described primary reaction (Fig. 1, A table), elementary probe of described wild-type and described INVADER oligonucleotide are one in front and one in back hybridized in described target nucleic acid to form overlay structure.Unpaired " flap " is included in 5 ' end of the elementary probe of described WT.Structure specific enzymes (for example CLEAVASE enzyme, the 3rd ripple science and technology) identification is described overlapping, cuts away described unpaired flap, and it is released to the target-specific product.In described secondary reaction, the product of this cutting plays INVADER oligonucleotide on WT FRET (fluorescence resonance energy transfer) (WT-FRET) probe, to produce the described structure (A table) of structure specific enzymes identification once more.When described two dyestuffs on the single FRET probe are separated by cutting (pointing out), be created in the detectable fluorescent signal on the background fluorescence by the arrow among Fig. 1.Therefore, the cutting of this secondary structure causes the increase of fluorescence, indication WT allelotrope (perhaps mutation allele, if described test for mutant allele produces detectable signal and dispose) existence.In some embodiments, detection at each allelotrope or locus, provide have different markers the FRET probe (for example, by emission or excitation wavelength different distinguishable, perhaps detect distinguishable by time resolved fluorescence) so that can detect different allelotrope or locus in the single reaction.In such embodiments, described elementary probe combinations (set) can be joined together in single test with described different FRET probe, allows the comparison from the described signal of each allelotrope or locus in the same sample.

If elementary probe oligonucleotides and target nucleotide sequences are not Perfect Matchings (for example, resembling elementary probe of MT and WT target, Fig. 1, B table) at cleavage site, described eclipsed structure does not form and cutting is suppressed.Compare with non-overlapped structure, the described structure specific enzymes of use (for example, CLEAVASEVin enzyme, the 3rd ripple science and technology) cuts described eclipsed structure (for example at least 340 times) more efficiently, allows described allelic fabulous differentiation.

Described probe need not the many signals (that is, linear signal amplification) of temperature cycle running (turn over) to produce each target.Similarly, each target-specific product can allow the cutting of many FRET probes.

Elementary INVADER test reaction directed target DNA to be detected (or RNA).Described target DNA is a restricted component in the cutting of first invasive, because described INVADER and elementary probe provide with molar excess.In the cutting of second invasive, restrictive is the flap that discharges.When these two cleavage reactions carry out in succession, accumulate according to the amount linearity of target DNA from the fluorescent signal of hybrid reaction (compositereaction).

In certain embodiments, described INVADER test or other Nucleotide detect to test and use the site design oligonucleotides and/or the bridge joint oligonucleotide that can get to carry out.Such method, program and composition be at United States Patent (USP) 6,194, and 149,6,358,691,6355,437, among U.S. Patent Application Serial Number 09/882,945 and PCT application WO9850403 and the WO0198537 description is arranged, all patents are complete specifically to be quoted as a reference.

In some preferred embodiments, if target sequence is present in the sample, described sample is exposed to oligonucleotide and reagent, comprise described sample is exposed to oligonucleotide and reagent that under the condition that forms the invasive cutting structure between described target sequence and the described oligonucleotide wherein said invasive cutting structure is cut by described cutting reagent and forms cleaved products.

In some embodiments, described target sequence comprises first area and second area, described second area is close in the downstream of first area and with it, described oligonucleotide comprises first and second oligonucleotide, wherein the part of described at least first oligonucleotide is complementary to the first part of target sequence fully, wherein said second oligonucleotide comprises 3 ' part and 5 ' part, and wherein 5 ' part is complementary to the second section of described target nucleic acid fully.

In some preferred embodiments, if target sequence is present in the sample, described sample is exposed to oligonucleotide and reagent, comprise described sample is exposed to oligonucleotide and reagent that under the condition that forms the invasive cutting structure between target sequence and the oligonucleotide wherein said invasive cutting structure is cut by described cutting reagent and forms cleaved products.

In some preferred embodiments, if described target sequence is present in the described sample, described sample is exposed to oligonucleotide and reagent, comprise described sample is exposed to oligonucleotide and reagent under the condition that forms the invasive cutting structure between described target sequence and the described oligonucleotide, wherein said invasive cutting structure is cut the reagent cutting and forms cleaved products.

In some particularly preferred embodiments, target sequence comprises first area and second area, described second area is close in the downstream of described first area and with it, and described oligonucleotide comprises first and second oligonucleotide, wherein the part of described at least first oligonucleotide is complementary to the described first part of described target sequence fully, wherein said second oligonucleotide comprises 3 ' part and 5 ' part, and wherein said 5 ' part is complementary to the described second section of described target nucleic acid fully.

In certain embodiments, the invention provides the test kit that uses INVADER detection reagent (for example, elementary probe, INVADER probe and FRET box) to analyze the sample (for example cell lysate of blood sample of He Binging or merging) of merging.In preferred embodiments, described test kit further comprises the specification sheets that how to carry out described INVADER test, and in some embodiments, how described INVADER being detected test is applied to from the sample of the merging of many individualities or from the specification sheets of " merging " samples of a lot of cells (for example, from biopsy samples) of single research object.

The present invention further provides such test, wherein hybridizing with many wheels of oligonucleotide probe and do not needing the use temperature circulation (promptly, for the periodic sex change of target nucleic acid chain) or nucleic acid synthetic (promptly, in the cutting process of probe the substituting based on polymerization for target or probe nucleic acid chain), target nucleic acid is re-used or recirculation.When cleavage reaction (for example substitutes by probe-probe probe is placed condition on the target chain by type discharge under, or by the balance between probe/target combination and the separation, perhaps by comprising the associating of these mechanism, (people such as Reynaldo, molecular biology magazine (J.Mol.Biol.) 97:511-520 (2000)) when carrying out, multiple probe can be hybridized in identical target, allows the generation of multiple cutting and multiple cleaved products.

INVADER tests reaction:

In the preferred embodiment of INVADER DNA test, two oligonucleotide (the elementary probe of distinguishing and INVADER oligonucleotide) are hybridized in target DNA in tandem to form overlay structure.5 ' end of described elementary probe comprises the 5 ' flap (Fig. 1) of not hybridizing in target DNA.3 ' Nucleotide of bonded INVADER oligonucleotide is overlapped in described elementary probe, but does not need to hybridize in target DNA.The CLEAVASE enzyme is discerned this overlay structure, and unpaired 5 ' flap cuts down with described elementary probe, and it is discharged as the target-specific product.Described elementary probe is through designing to have the melt temperature close with temperature of reaction.Therefore, under the equitemperature test conditions, the excessive elementary probe that provides is in the target DNA cocycle.This allows at the elementary probe cutting of many wheels of each target DNA, and the amplification of the 5 ' flap quantity that discharges.

In secondary reaction, 5 ' flap of each release can play the work of INVADER oligonucleotide on FRET (fluorescence resonance energy transfer) (FRET) box in order to produce the overlay structure (Fig. 1) that another one is discerned and cut by the CLEAVASE enzyme.When the FRET box was cut, fluorophore (F) and quencher (Q) were separated, and produce detectable fluorescent signal.With initial reacting phase seemingly, 5 ' flap of release and the circulation of FRET box cause the fluorescent signal that increases.Described carrying out simultaneously in identical hole with secondary reaction at first.

The binary form of described INVADER test has been realized detecting in two kinds of dna sequence dnas in single hole.The most frequently, this comprises the detection (for example, among the miRNA) of two varients of specific polymorphism.Described binary form uses two kinds of elementary probes of different distinguishings, and every kind has 5 unique ' flap, the FRET box different with two kinds, and every kind has the distinct fluorophore of wave spectrum.By design, 5 ' flap of release will only be incorporated into their FRET boxes separately to produce the signal of target-specific.

In some embodiments, the invention provides to comprise and carry out one or more components required in this invention.For example, the invention provides and store or send enzyme of the present invention and/or carry out the essential reactive component of cutting test (for example, described INVADER test).By way of example, be not to anticipate test kit of the present invention to be restricted to the associating of any special configuration or component, following chapters and sections have been described an embodiment carrying out test kit of the present invention:

In some embodiments, the reagent below test kit of the present invention provides:

The elementary oligonucleotide of CLEAVASE enzyme

DNA reaction buffer I INVADER oligonucleotide

FRET box 1 (for example, F)

FRET box 2 (for example, R)

The mutant DNA contrast

Wild-type DNA contrast

The blank of " no target "

In other embodiment, test kit of the present invention is configured for the direct detection of RNA.These test kits can provide following reagent:

The elementary probe of CLEAVASE enzyme

Oligonucleotide

DNA reaction buffer I INVADER oligonucleotide

FRET probe 1 (for example, F)

FRET probe 2 (for example, R)

Secondary reaction target 1

Secondary reaction target 2

ARRESTOR oligonucleotide 1

ARRESTOR oligonucleotide 2

The mutant DNA contrast

Wild-type DNA contrast

The blank that " does not have target "

Extra consideration must be arranged, and it is relevant with the non-expectation function that is produced by the particular combinations of oligonucleotide in the single reaction.The generation of the target independence that a kind of such effect is a background signal.Some oligonucleotide and the combination of other oligonucleotide are not having can produce signal in the presence of the special target to be detected in described INVADER test.The combination of these oligonucleotide is separated into different Chi Zhongneng is used to alleviate this effect.Similarly, the combination of some oligonucleotide can phonily suppress to produce from the signal of desired target.Again, these combinations are separated in the different ponds and can alleviate this effect.

Designing probe combination (set) (for example, oligonucleotide and/or its sequence) is to be suitable for the use in the miRNA detection test, and it utilizes reaction design and optimized guilding principle (for example see, test chapters and sections) provided herein.For example, in some embodiments, reduce described temperature of reaction (for example, to 50-60 ℃) to carry out the hybridization of zonule.

In some embodiments, test kit of the present invention provides in order to carry out the tabulation (for example, reagent, supplies and/or equipment) of method of the present invention by the additional component of user's supply.For example, and be not to be intended to the tabulation of extra like this component is limited to any special component, an embodiment of such tabulation has comprised as follows:

Transparent CHILLOUT-14 liquid wax (MJ Research) or do not have RNase optical grade mineral oil (Sigma, Cat.No.M-5904)

96 hole polypropylene trace flat boards (MJ Research, Cat.No.MSP-9601)

Aseptic 1.5ml or 2.0ml Eppendorf tube

Disposable pneumo colloidal sol aseptic, no DNase/RNase is isolated the pipettor suction nozzle

Multichannel pipettor (0.5-10 μ l, 2.5-20 μ l)

Thermocycler or other thermals source (for example, lab oven at or heat block)

Laboratory equipment miscellaneous (test-tube stand, micropipet, multichannel pipettor, Eppendorf centrifuge, shaker mixer)

Fluorescence trace plate reader (preferred plate reader be from top reading and be equipped with have below the spectral filter of feature:

Excitation-emission

(wavelength/bandwidth) (wavelength/bandwidth)

485nm/20nm 530nm/25nm

560nm/20nm 620nm/40nm

In some embodiments, test kit of the present invention provides method for convenience of the present invention to carry out the tabulation (for example, reagent, supplies and/or equipment) of the additional component of being supplied with by the user.For example, and be not to be intended to so optional component tabulation is limited to any special component, an embodiment of such tabulation has comprised as follows:

Aseptic 8 test tube bars or trace dull and stereotyped (optionally)

Disposable plastic tank (optionally)

Dull and stereotyped Sealing strap (optionally)

In some embodiments, the tabulation of the essential component that the carrying out that test kit of the present invention provides the inventive method for convenience supplied with by the user, a plurality of for this reason optional things are acceptable (for example sample preparation reagents boxes).For example, and be not to be intended to the tabulation of such selectable components is limited to any special component, an embodiment of such tabulation comprises as follows:

QIAGENQI AMP blood test kit

Gentra Systems PUREGENE test kit

Gentra Systems GENERATION product

In some embodiments of test kit, provide the detailed experiments scheme.The experimental program (for example, the preferable procedure of preparation and manufacturing reaction mixture) of the compilation of INVADER test reaction is provided in preferred embodiments.In particularly preferred embodiments, the experimental program of the compilation of reaction mixture comprises the help of computer or drawing, (for example to reduce risk that the inventive method makes a mistake in carrying out, make things convenient for the form of calculating of the volume of the reagent that multiple reaction needs, and the slab design that auxiliary configuration contains the porous test panel of many tests reactions instructs).

In some embodiments, provide append file, for example subsidized the testing program of program, for example for the preparation of additional agents, or in order to be used for the experimental program of the inventive method specimen preparation.In preferred embodiments, append file comprises that guilding principle and preventive measures that non-technical personnel for convenience or inexperienced user provide the successful use of described method and reagent tabulate.In particularly preferred embodiments, append file comprises the guide of dealing with problems, and for example, has described the possible problem that the user meets with, and provides and be intended to help the user to solve or avoid such problem and the solution that proposes or the guide of correcting method.

In preferred embodiments, sample is diluted to the concentration that adds 10 μ l corresponding to each reaction.The concentration of 100ng sample should be 15ng/ μ l.

B. rolling-circle replication

In other embodiment, (NJ) detection that is used to the miRNA detection architecture (is for example seen United States Patent (USP) 6,344 to the rolling-circle replication method for Amersham Biosciences, Piscataway, 329,6,143,495,6,316,229,6,210,884,6,183,960 and 6,235,502; They each is incorporated herein by reference).In some embodiments, rolling-circle replication is used to detect the ring-type miRNA detection architecture that produces from the annealing with the single oligonucleotide end of miRNA annealed.In some embodiments, the end of oligonucleotide is hybridized in there not being eclipsed miRNA.This oligonucleotide is having or is not having in the presence of the miRNA and can be connected.Yet, described ligation have in the presence of the described miRNA more efficient.In such embodiments, will compare with the control reaction that lacks miRNA along with the ring molecule level of time detecting.

In other embodiment, the end of described oligonucleotide is hybridized in the miRNA that overlapping end is arranged to produce the invasive cutting structure.Such structure was cut before connecting, thereby improved the specificity that described ring-type detection architecture produces.

Rolling circle amplification (RCA) comprises duplicating of single-stranded cyclic DNA molecule.In RCA, the primer hybridization of rolling-circle replication is to use chain to substitute the rolling-circle replication of the nucleic acid molecule of archaeal dna polymerase in the circular nucleic acid molecule afterwards.Described amplification occurs among the rolling-circle replication of single reaction cycle.Rolling-circle replication obtains containing the big dna molecular of the tandem repetitive sequence of nucleotide sequence.This dna molecular is called as tandem sequence DNA (TS-DNA).

In some embodiments, utilize the rolling circle amplification (LM-RCA) that duplicates the connection mediation that comprises attended operation before.In described attended operation, probe hybridization is the covalence closed single-chain nucleic acid of being connected to form of hybridization probe end in its similar target nucleic acid sequence (if present) afterwards.After the connection, the rolling-circle replication primer hybridization is to use chain to substitute the rolling-circle replication of the ring molecule of archaeal dna polymerase in probe molecule afterwards.Usually, LM-RCA comprises open loop probe and target sample mix, obtain probe-target sample mixture, and promoting to hatch described probe-target sample mixture under the condition of hybridizing between described open loop probe and the target sequence, ligase enzyme is mixed with described probe-target sample mixture, obtain connecting mixture, and promoting to hatch described connection mixture under the condition that described open loop probe connects to form amplification scoring ring (ATC), rolling-circle replication primer (RCRP) is mixed with the described mixture that is connected, obtain primer-ATC mixture, and promoting to hatch described primer-ATC mixture under the condition of hybridizing between described amplification scoring ring and the described rolling-circle replication primer, archaeal dna polymerase is mixed with described primer-ATC mixture, obtain polysaccharase-ATC mixture, and promoting to hatch described polysaccharase-ATC mixture under the condition of described amplified target circle replication, amplification scoring ring described here duplicate the formation that causes tandem sequence DNA (TS-DNA).

C. extra detection method

The invention is not restricted to the INVADER test or roll the ring test detection.The method of the detection of any permission miRNA detection architecture can be utilized.As example, find that the unrestriced detection test that is used for the inventive method has description below.

1. cross experiment

In some embodiments of the present invention, detection architecture uses cross experiment to detect.In cross experiment, have or the existence of not having a specific nucleic acid sequence is measured based on the ability of hybridizing from the DNA of sample in complementary DNA molecule (for example, oligonucleotide probe).It is utilizable using the various cross experiments of various hybridization and detection technique.The test select be described in below provide.

A. Za Jiao direct detection

In some embodiments, the hybridization of probe and aim sequence (for example, SNP or sudden change) is by manifesting bonding probes (for example, Northern or Southern test; For example see people (eds.) such as Ausabel, molecular biology experimental program (Current Protocols inMolecular Biology) in the present age, John Wiley ﹠amp; Sons, NY[1991]) directly detect.In these trials, genomic dna (Southern) or RNA (Northern) separate from research object.Described DNA or RNA cut with near the Restriction Enzyme that does not rarely cut any mark to be analyzed in the series of genes group then.Described DNA or RNA are separated (for example, on the sepharose) then and are transferred on the film.(for example, by the introducing a radioactive nuleus thuja acid) probe of mark or the probe that is specific to described SPN to be detected or sudden change be allowed to low, in or contact with described film under the high stringent condition.Unconjugated probe is removed and the bonded existence detects by the probe that manifests described mark.

B. use the detection of the hybridization of " DNA chip " test

In some embodiments of the present invention, the varient sequence uses the test of DNA chip hybridization to detect.In this test, a series of oligonucleotide probes are attached to solid support.It is unique (for example, miRNA target sequence) that described oligonucleotide probe is designed to for specific target sequence.The target DNA sample contacts and hybridization is detected with described DNA " chip ".

In some embodiments, described DNA chip test is GeneChip (Affymetrix, Santa Clara, CA; For example see U.S. Patent number 6,045,996,5,925,525 and 5,858,659; They each quoted as a reference herein) test.Described GeneChip technology is used small-sized, the high density arrays of the oligonucleotide probe that invests " chip ".Probe array is produced by the light indication chemical synthesis of Affymetrix, and it has united the photolithography manufacturing technology that solid state chemistry is synthetic and semi-conductor industry adopts.Having used a series of photolithography egative films in order to define the chip exposed sites, is specificity chemosynthesis step afterwards, and described method has made up the high density arrays of oligonucleotide, and each probe predetermined position in array is arranged.Multiple probe array synthesizes on the sandwich wafer of big glass simultaneously.Described sandwich wafer is cut into square then, and independent probe array is packaged in the plastic injection magazine, and they avoid the influence of environment and as the chamber of hybridization described box protection.

Nucleic acid to be analyzed is separated, increases by PCR, and carries out mark with the fluorescence report group.The DNA of described mark uses fluid operating station (fluidics station) and described array to hatch then.Described array is inserted in the scanner then, detects the pattern of hybridization at this.Collect hybridization data, be the emission light of the fluorescence report group that is integrated into target, described target is incorporated into described probe array.Since the sequence of each probe and position are known on the described array, by complementarity, the identity that is applied to the target nucleic acid of described probe array can be determined.

In other embodiment, (Nanogen, San Diego CA) (for example see U.S. Patent number 6,017,696,6,068,818 and 6,051,380 to utilize the DNA microchip that contains the electronics capturing probe; They each quoted as a reference herein).By microelectronic use, the technology of Nanogen has realized arriving and from the active motion of the charged molecule of specified test site on the semiconductor microactuator chip with concentrate (concentration).For the particular target sequence be unique DNA capture probe be placed on by electronically or " being positioned " described microchip on the specificity site.Since DNA has very strong negative charge, it can be moved to the positive charge zone by electronically.

At first, test site or delegation's test site activate with the positive charge electronics on the described microchip.Secondly, the solution that contains described dna probe is introduced on the described microchip.The negative charge probe moves to positively charged site soon, and they concentrate and by the site of Chemical bond on described microchip at this.Clean described microchip then, another solution that adds distinct dna probe is finished in conjunction with the array of dna probe up to specificity.

Then specimen is hybridized the existence of analyzing target sequence in target sequence by measuring which described DNA capture probe.Elementary charge also is used to move and enriched target molecule one or more test site to the described microchip.The electron density of sample DNA has promoted the quick hybridization (hybridization can take place) of sample DNA and complementary capture probe in several minutes on each test site.In order to remove the DNA of any unconjugated or non-specific binding from each site, the polarity in described site or electric charge are reversed to electronegative, therefore force any DNA unconjugated or non-specific binding to get back to solution away from described capture probe.Fluorescent scanning instrument based on laser is used to detect combination.

In embodiment further, based on by capillary difference, (ProtoGene, Palo Alto CA) (for example see U.S. Patent number 6,001,311,5,985,551 and 5,474,796 to the fluidic array technique on the isolated plane (chip); They each quoted as a reference herein).The technology of Protogene can be isolated in the fact on the different plane of the surface tension that is given by chemical packs based on fluid.In case so isolated, oligonucleotide probe is directly synthetic on described chip by the spray ink Printing of reagent.The array that has the reaction site that is defined by surface tension is installed on the X/Y mobile platform under four piezo nozzles of a cover, and each nozzle is represented of four standard DNA bases.Described mobile platform moves and suitable reagent is delivered to each reaction site along every row of described array.For example, Aamidite only be delivered to amidite A among that synthesis step by link coupled site or the like.General reagent and scavenging solution are by covered with (flooding) whole surface and by centrifugal its removal is sent then.

Use the technology of Protogene to be attached to described chip for the unique dna probe of purpose target sequence (for example, miRNA target sequence).Described chip contacts with the goal gene of pcr amplification then.After the hybridization, unconjugated DNA is removed and hybridizes and uses any suitable method to detect (for example, the fluorescence by the fluorophor integrated goes cancellation).

Yet in other embodiment, " pearl test " is used to detection (Illumina, San Diego, the CA of polymorphism; For example see, open WO 99/67641 of PCT and WO00/39587, they each is incorporated herein by reference).Illumina uses the pearl array technique of having united fiber illuminated and oneself being assembled into the pearl of array.Each fiber illuminated depends on the diameter of described light beam and contains thousands of to millions of single fibers.The described pearl oligonucleotide bag quilt of the detection that is specific to specific SNA or sudden change.Several pearls are united the set that is specific to described array with formation.In order to test, described pearl test contacts with the sample (for example, nucleic acid samples) of the research object for preparing.Hybridization uses any suitable method to detect.

C. the enzyme process of hybridization detects

In some embodiments of the present invention, hybridization detects by the enzyme cutting of specificity structure.

In some embodiments, TaqMan test (PEBiosystems, Foster City, CA are used in the hybridization of bonding probes; For example see, U.S. Patent number 5,962,233 and 5,538,848, they each quoted as a reference herein) detect.Described test is carried out in the process of PCR reaction.Described TaqMan test has utilized 5 '-3 ' exonuclease activity of AMPLITAQ GOLD archaeal dna polymerase.The probe that is specific to specific allelotrope or sudden change is included among the described PCR reaction.Described probe is made up of the oligonucleotide that 5 ' reporter gene dyestuff (for example, fluorescence dye) and 3 ' quencher dyes are arranged.In the PCR process, if described probe is incorporated into its target, 5 '-3 ' nucleic acid hydrolysis activity of AMPLITAG GOLD polysaccharase has been cut the probe between described reporter gene and the described quencher dyes.The reporter gene dyestuff separately causes the increase of fluorescence from quencher dyes.Described signal is accumulated along with each circulation of PCR and can be monitored with luminoscope.

In embodiment further, polymorphism is used the test of SNP-IT primer extension (Orchid Biosciences, Princeton, NJ; For example see, U.S. Patent number 5,952,174 and 5,919,626, they each is incorporated herein by reference) detect.In this test, SNP differentiates by using specificity synthetic dna primer and archaeal dna polymerase to extend described DNA chain by a base selectivity of suspicious SNP position.DNA in the purpose zone is amplified and sex change.Polymeric enzyme reaction uses the miniaturized system that is called as microfluid to carry out then.Detection is finished by marker being joined the Nucleotide in the target sequence position under a cloud.Described marker is integrated into described DNA can detect (for example, if described nucleic acid contains the biotin labeling thing, detection can be via the fluorescent-labeled antibody that is specific to vitamin H) by any suitable method.

2. other detects test

Extra detection useful in the detection of miRNA detection architecture is tested, and includes but not limited to enzyme mispairing cutting method (for example, Vairagenics, U.S. Patent number 6,110,684,5,958,692,5,851,770, complete quoting as a reference) herein; The polymerase chain reaction; Branch's hybridizing method (for example, Chiron, U.S. Patent number 5,849,481,5,710,264,5,124,246 and 5,624,802, complete herein quoting as a reference); NASBA (for example, U.S. Patent number 5,409,818, complete herein quoting as a reference); Molecular beacons technology (for example, U.S. Patent number 6,150,097, complete herein quoting as a reference; Electronic sensor (Motorola, U.S. Patent number 6,248,229,6,221,583,6,013,170 and 6,063,573, complete herein quoting as a reference); Circle probe technology (for example, U.S. Patent number 5,403,711,5,011,769 and 5,660,988, complete herein quoting as a reference); DadeBehring signal amplification method (for example, U.S. Patent number 6,121,001,6,110,677,5,914,230,5,882,867 and 5,792,614, complete herein quoting as a reference); Ligase chain reaction (LCR) (Barnay, Proc.Natl.Acad.Sci USA 88,89-93 (1991)); And sandwich hybridization method (for example, U.S. Patent number 5,288,609, complete herein quoting as a reference).

Experiment

In order to prove and further to illustrate some embodiment preferred and all respects of the present invention, the following examples are provided, and are not interpreted as limiting the scope of the invention.

Embodiment 1

Material and method

Following ultimate density (unless having dated) is used for all reactions:

Probe=1 μ M

INVADER＝1μM

ARRESTOR＝2.67μM

CLEAVASE XII enzyme=30ng

All synthetic miRNA oligonucleotide are from the Dharmacon purchase and at 20% denaturing acrylamide gel purifying.Synthetic miRNA is used to measure the suitableeest (face as follows) and the LOD of temperature.

INVADER, probe and ARRESSTOR oligonucleotide are synthetic by dna integration technology (IDT) or the 3rd ripple science and technology, carry out purifying on 20% denaturing acrylamide acid amides, unless point out in addition.

Following 2.5 * primary reaction damping fluid is used for (unless having dated in addition) all reactions:

25mM MOPS pH7.5

62.5mM ^KCl

0.125％Tween 20

0.125％Nonidet NP40

62.5mMMgSO ₄

5％PEG

Unless point out in addition, all first heat that are reflected at are covered by 10 μ l mineral oil before hatching.

Unless have datedly in addition, synthetic miRNA contains 5 ' hydroxyl.Relatively the experiment of 5 ' phosphorylation and non-phosphorylating synthetic miRNA target detection is pointed out, the ability that the INVADER test detects these two dissimilar synthetic molecules does not have significantly difference.

Embodiment 2

The temperature optimization experiment of let7 and mir-1

The oligonucleotide design of let-7 is presented among Fig. 5.The oligonucleotide design of mir-1 is presented among Fig. 5.Below elementary mixture be made into and in 96 orifice plates, ℃ hatched 30 minutes in 50C ± 10.In addition, there is not the overall mixture (master mix) of target to be produced (add entry and replace RNA).All reactions cover to stop evaporation with mineral oil.

The primary reaction component	Stock concentration	The amount that adds
			The primary reaction damping fluid	2.5X	4μl
Probe oligonucleotides is (for let 7SEQ ID NO:2,6 and 9; For miR-1SEQ ID NO:12,16 or 19)	10μM	1μl
			The INVADER oligonucleotide is (for let7 SEQ ID NO:1,5 and 8; For miR-1 SEQ ID NO:11,15 or 18)	10μM	1μl
CLEAVASE IX or XII enzyme	40ng/ μ l CLEAVASE IX enzyme or 60ng/ μ l CLEAVASE XII enzyme	0.5μl
			tRNA	20ng/μl	1.5μl
Synthetic miRNA is (for let-7a SEQ ID NO:4; For miR-1 SEQ ID NO:14)	100pM	2μl
			Summation		10μl

After primary reaction is finished, add the secondary reaction mixture below the 5 μ l, and described reaction was hatched 10-15 minute 60 ℃ of reactions then.

The primary reaction component	Stock concentration	The amount that adds
			Water (or damping fluid of CLEAVASE IX enzyme test)		2μl
FAM FRET probe (SEQ ID NO:21)	10μM	1μl
			The primary reaction target is (for let-7 SEQ ID NO:22; For miR-1 SEQ ID NO:40)	1.5μM	1μl
The ARRESTOR oligonucleotide is (for let-7 SEQ ID NO:3,7 or 10; For miR-1SEQ ID NO:13,17 or 20)	40μM	1μl
			Summation		5μl

After described reaction was finished, described flat board used the excitation wavelength of 485nm and the emission wavelength of 530nm to carry out reading in CYTOFLUOR4000 fluorescence trace flat bed reader.The result is presented in Fig. 6 and 7.When 3 ' end of described INVADER oligonucleotide be 2 '-oxygen-methylated the time, 5 of described INVADER oligonucleotide ' end is enhanced to terminal the piling up of 3 of described miRNA '.In addition, 2 of described INVADER oligonucleotide 5 ' end '-oxygen-methylating has increased temperature of reaction.To such an extent as to extend 2 of INVADER oligonucleotide '-oxygen-methylated base they with the base pairing of initial two bases of described miRNA (SEQ ID NO:8 (1496-96-02) is to SEQ ID NO:23 (1496-96-03) in SEQ ID NO:9 (1496-96-OlR) design of let-7a), the optimum temperuture that has increased described reaction does not still strengthen described detection.

Embodiment 3

The LOD test of let-7 and miR-1

After measuring the peak optimization reaction temperature of the probe of each combination and INVADER oligonucleotide and measuring best work design (from the optimum net signal of temperature), following experiment is configured to use synthetic RNA to measure the LOD of described design.Following reaction mixture is dispensed into each hole and contains (face is dull and stereotyped as follows is provided with) in the 96 following orifice plates:

Component	Deposit concentration	The amount that adds
			The primary reaction damping fluid	2.5X	4μl
Probe for let-7 SEQ ID NO:6 for miR-1 SEQ ID NO:16 or 19	10μM	1μl
			The INVADER oligonucleotide for let-7 SEQ ID NO:5 for miR-1 SEQ ID NO:15 or 18	10μM	1μl
CLEAVASE XII enzyme	60ng/μl	0.5μl
			tRNA	20ng/μl	1μl
Summation		7.5μl

2.5 the setting below the miRNA concentration below the μ l is used adds with three duplicate samples or four duplicate samples:

＜--------[miRNA]-----→

	1nM	100pM	10pM	1pM	100fM	10fM	H2O
								A
B
								C
D

Described flat board covers (10 μ l) with mineral oil and hatched 2 hours at 50 ℃.After primary reaction was finished, the component below the 5 μ l joins each hole and described flat board was hatched 1.5 hours at 60 ℃.Described flat board carries out reading with above-described setting (seeing embodiment 2).

Component	Store concentration	The amount that adds
			Water (or damping fluid of CLEAVASE IX enzyme reaction)		2μl
FAM FRET probe (SEQ ID NO:21)	10μM	1μl
			Secondary target is (for let-7 SEQ ID NO:22; For miR-1 SEQ ID NO:40)	1.5μM	1μl
The ARRESTOR oligonucleotide is (for let7 SEQ ID NO:7; For miR-1 SEQ ID NO:17 or 20)	40μM	1μl
			Summation		5μl

Then the LOD of let-7 and mir-1 detects on the human rna sample.Above-described experimental program is utilized.(Clonetech, Palo Alto CA) is used the total people RNA sample of 50-100ng tissue specificity.The result is presented in Fig. 8 and 10.Use the described let-7a INVADER of total RNA test to detect as the in the past observed identical distribution expression pattern (profile) of tissue-derived let-7a expression level (people such as Pasquinelli, 408:86[2000]) that depends on.

Embodiment 4

The cross reaction experiment of Let-7a, c, e and f

This embodiment has described at the analysis for the cross reactivity of another hypotype of the probe of a let-7 hypotype and/or INVADER oligonucleotide.Adopt the experimental program of the synthetic let-7a setting of describing among the embodiment 3.Fig. 5 has shown the oligonucleotide design.Flat board setting below adopting:

		10nM	1nM	100pM	10pM	1pM	100fM	10fM	H 2O
												1	2	3	4	5	6	7	8
Let 7A	A
										Let 7A	B
Let 7C	C
										Let 7C	D
Let 7E	E
										Let 7E	F
Let 7F	G
										Let 7F	H

The result is presented among Fig. 9.For let-7a design, identical when described miRNA length, when a sequence change being arranged away from described cleavage site, the cross reactivity maximum.In other words, when mismatching more away from described cleavage site (let-7c), INVADER oligonucleotide/mismatching of miRNA hybridization zone obtains high cross reactivity.When base changed on the opposite of described cleavage site (or approaching with it), cross reactivity was minimum.For let-7a, the most serious cross reactivity is and let-7c that it has obtained 25% described signal.This embodiment has proved that described INVADER test can distinguish closely similar miRNA.

Embodiment 5

CLEAVASE IX enzyme is to CLEAVASE XII enzyme

This embodiment has described the optimization of the CLEAVASE enzyme that is used for the miRNA test.The testing program of above-described temperature optimization is utilized.Use 20ng CLEAVASE IX enzyme (the 3rd ripple science and technology, Madison, WI) or the CLEAVASE XII enzyme of 30ng (the 3rd ripple science and technology, Madison, WI).Following damping fluid is used for CLEAVASE IX enzyme:

2.5X primary reaction damping fluid: 25mM MOPS pH7.5,250mM KCl, 0.125%Tween 20,0.125% Nonidet NP40,31.25mM MgSO ₄, 10%PEG.

7.5X secondary reaction damping fluid: 87.5mM MgSO ₄

Following damping fluid is used to CLEAVASE XII enzyme:

2.5X primary reaction damping fluid: 25mM MOPS pH7.5,62.5mM KCl, 0.125%Tween 20,0.125% Nonidet NP40,62.5mM MgSO ₄, 5% PEG.

7.5X secondary reaction damping fluid: water

The experimental program of LOD experiment uses with CLEAVASE IX or XII enzyme.Measured the LOD of two enzymes.The result is presented among Figure 11.

Signal linear increase when analyzing along with the increase of let-7miRNA amount with CLEAVASE IX enzyme or CLEAVASE XII enzyme.Yet, R ²Value is bigger at CLEAVASE XII enzyme, has pointed out bigger linear relationship.In addition, described LOD is lower at CLEAVASE XII enzyme.2.5 the detection net signal of Ah's mole (net signal) is 20 countings and is 66.75 at CLEAVASE XII enzyme at CLEAVASE IX enzyme.

Embodiment 6

MiR-135, GAPDH and U6 RNA

A. detect the design of the oligonucleotide of miR135

This embodiment has described the mensuration of miRNA miR135 test design and LOD.The experiment of miR-1 such as embodiment 2 and 3 carry out with describing.Described oligonucleotide design has description in Fig. 5.For each described design (A-D) of detection of mir-135miRNA has utilized different INVADER and probe oligonucleotides.Relatively all described designs temperature optimization result of experiment of carrying out is presented among Figure 13.Design D produces the highest signal.The LOD result of experiment of use-testing design D is presented among Figure 14.

Figure 14 A has presented the thick counting that produces from four revision tests at each target level of pointing out.The average counter that each target level obtains is represented net signal and is multiple (FOZ) on zero.The detectability of miR-135 target is 164 zmoles in this experiment, is equivalent to 98,743 molecules.Figure 14 B contains the diagram of the average counter that obtains in each concentration, and points out that the INVADER test is linear in many concentration ranges of test.

B. detect the design of the oligonucleotide of GAPDH and U6 RNA

In some cases, the common detection generally expected that with the RNA that one or more miRNA kinds exist described RNA expresses in tissue-specific mode with constant level during for example binary was tested in all cells.Therefore the INVADER test is designed to generally two distinct RNA of visible in all cells type: the RNA of people's glyceraldehyde 3-phosphate dehydrogenase (hGAPDH) and U6.

Under the situation of hGAPDH, following oligonucleotide is used to binary miRNA and detects test: INVADER oligonucleotide (SEQ ID NO:41); Probe (SEQ ID NO:42); ARRESTOR oligonucleotide (SEQ ID NO:43); SRT oligonucleotide (SEQ IDNO:49), FRET oligonucleotide (orchil) (SEQ IN NO:48).

Under the situation of U6, belong to (arabidopsis) to mouse to mustard to human 8 different types of U6 RNA sequence alignments from beautiful new rhabditis axei, be suitable for the zone of " general " INVADER test design with evaluation.Described comparison is presented among Figure 12; Generation is SEQ ID NO:93-95 with the oligonucleotide sequence that detects this sequence.

Proved that in the initial trial of using SEQ ID NO:45-47 and these oligonucleotide to carry out on the cell lysate signal from the U6 reaction reached very saturated before the miRNA signal, may be because the interior a large amount of U6 RNA of cell.Therefore, carry out drop reaction, whether can make this probe combinations be suitable for the INVADER of final concentration scope from 1 μ M to 12.5nM and the binary miRNA detection test of probe with the concentration of determining dilution probe and INVADER oligonucleotide.12.5-50nM between described INVADER and the final concentration of the probe oligonucleotides binary miRNA that is suitable for miR-1d and let-7a detect.ARRESTOR, SRT and FRET concentration and probe concentration are as describing among the embodiment in front.The detection that further experimental results show that the U6RNA that uses " general " U6RNA oligonucleotide (SEQ IN NO:93-95) is with suitable with the detection of SEQ ID NO:45-47.

Embodiment 7

The detection of let-7, GADPH and U6 RNA in the cell lysate

A. the detection of let 7a in the cell lysate

This embodiment has described directly in total cell RNA and from human osteosarcoma cell line MG63 (the 3rd ripple science and technology, Madison, WI; Catalog number (Cat.No.) CRL-1427) detection of let-7 miRNA in the not inductive inoblast.Total cell RNA, as former description ground (people such as Chomczynski, analytical biochemistry (Anal.Biochem.) 162:156-156 (1987)), use TRIZOL (Gibco-BRL) to extract, and people such as cell lysate such as Eis, Nature Biotechnol (Nature Biotechnology), 19:673-6 (2001) is prepared; Publish an article for two and be incorporated herein by reference.

React following the setting.(the people such as Eis of the synthetic miRNA target of concentration shown in 5 μ l cell lysates of five equilibrium, the lysis buffer being, Nature Biotechnol (NatureBiotechnology), 19:673-6 (2001)) or 5 μ l 20ng/ μ l tRNA (as no target contrast) join in the dull and stereotyped suitable hole of microtitration with pipettor.The overall mixture of primary reaction is made into to be used for 96 reactions, contains following reagent.

Reagent	Stock concentration	The amount of each reaction (μ l)	Join the total amount (μ l) of overall mixture
				Probe mixture oligonucleotide 1496-78-01R (SEQ ID NO:6) and INVADER oligonucleotide 1496-78-02 (SEQ ID NO:5)	Probe 20 μ M/INVADER oligonucleotide 200 μ M	0.5	45
CLEAVASE XII enzyme	60ng/μl	0.5	45
				Elementary damping fluid	2.5X	4	360
Summation		5	450

The aliquot of the overall mixture of 5 μ l primary reactions joins in the hole of containing suitable target or contrast.Described flat board covers with mineral oil (10 μ l) and hatched 2 hours at 53 ℃.After primary reaction was finished, the following component of 5 μ l joined in each hole, and described flat board was hatched 1.5 hours at 60 ℃.Reading (seeing embodiment 2) is carried out in the above-described setting of described dull and stereotyped use.

The secondary reaction component	Stock concentration	The amount that adds
			Water (or damping fluid of CLEAVASE IX enzyme test)		2μl
FAM FRET probe (SEQ ID NO:21)	10μM	1μl
			Secondary reaction target (SRT) SEQ ID NO:22	1.5μM	1μl
ARRESTOR oligonucleotide SEQ ID NO:7	40μM	1μl
			Summation		5μl

All targets are tested with four duplicate samples.Total RNA is plotted among Figure 15 with the average counter that different cell count obtained that cell lysate is tested.The typical curve that obtains from the INVADER test of the synthetic let-7amiRNA of known quantity be used to the to extrapolate let-7a copy number of each cell.As people such as Eis, measures in the Nature Biotechnol (Nature Biotechnology), 19:673-6 (2001) in the vaccine program process of quantity before lysis of the cell of generation cell lysate with describing, quote as a reference herein.In this experiment, the total RNA extract that obtains from 156 cells, reached the detectability of cell lysate.

B. there is not Mg ⁺⁺The cracking that exists

Optionally the cracking program is carried out as follows.When having pointed out the cracking program above using, long mRNA promptly from GADPH, does not detect the amount of expection.Carry out described experiment to check Mg ⁺⁺Effect to RNA extraction in the lysate.There is being or do not having MgCl ₂Existing down, the cracked extract compares with using the total cell RNA extract that uses TRIZOL to prepare as described above.

Hela cell (7.5 * 10 ⁶Cell) is suspended in the 10mM MOPS buffered soln that has 100mM KCl of 100 μ l pH7.5.10 μ l aliquots join (separate) separately in vitro and two different lysis buffer cracking that are prepared as follows with 100 μ l:

The MOPS lysate has Mg ++	The MOPS lysate does not have Mg ++
		180μl 11μg/ml tRNA	180μl 11μg/ml tRNA
0.5ml NP40	0.5ml NP40
		4ml 0.5M MOPS	4ml 0.5M MOPS
0.5ml 1M MgCl 2	N/A
		4.82ml water	5.32ml water
10ml	10ml

All test tubes are hatched 15 minutes with lysing cell at 80 ℃ then, centrifugal then collection fragment.The following INVADER that joins of the aliquot of various lysate 5 μ l reacts.

Elementary INVADER reaction is described as top let-7a; The PI oligonucleotide mixture of GADPH (SEQ ID NO:41-43) and U6 (SEQ ID NO:45-47 is at the 50nM final concentration) also is made into.

Component	The amount that each reaction adds	Final concentration
			The PI oligonucleotide mixture *	0.25μl	Each 1 μ M *
Water	0.25μl	0.25μl
			CLEAVASE XII enzyme 60ng/ μ l	0.5μl	0.5μl
	4μl	5μl
			Summation	5μl	5μl

^*Elementary INVADER reaction is described as top let-7a; The PI oligonucleotide mixture of GADPH (SEQ ID NO:41-43) and U6 (SEQ ID NO:45-47 is at the 50nM final concentration) also is made into.

The primary reaction mixture was hatched 1 hour at 49 ℃.Below the aliquot of second reaction mixture be added into then:

Component	The amount that each reaction adds
		The secondary reaction mixture *	1.5μl
Water	3.5μl
		Summation	5μl

The secondary reaction mixture be included in concentration as shown in embodiment 7A SRT (for let-7, SEQ ID NO:22; For GADPH and U6, SEQ ID NO:49) target, the FRET oligonucleotide (for let-7, SEQ ID NO:21; For GADPH and U6, SEQ ID NO:48), arrestors (for let-7, SEQ IN NO:7; For GAPDH, SEQ ID NO:43 and for U6, SEQ ID NO:47).

Secondary reaction was carried out 1 hour at 60 ℃.Be reflected at as the CYTOFLUOR trace plate reader of describing among the embodiment 2 and carry out reading.The result is presented among Figure 16, and the existence that shows the GAPDH signal depends on does not have Mg in the lysis buffer ⁺⁺Existence, and U6 RNA signal is no matter have or not Mg ⁺⁺Existence all keep constant relatively.Extra experiment confirm, all RNA with no Mg ⁺⁺Obtaining those RNA in the cracking that exists is can be detected in total cell RNA of level quite.

Embodiment 8

Multiple miRNA detects alternative INVADER test design

A.let-7A detects alternative design

This embodiment has described let-7a miRNA and has detected creation and the test that alternative oligonucleotide designs.In a series of experiment, produced optionally design of a cover, the target-specific zone of wherein said INVADER oligonucleotide and described probe oligonucleotides all is that 11 Nucleotide are long.Produced second Analysis of Nested Design, the target-specific zone of wherein said probe oligonucleotides is that 10 Nucleotide are long, and described INVADER oligonucleotide is that 12 Nucleotide are long.

1. oligonucleotide design

A.11 aggressiveness probe and INVADER oligonucleotide design

Fig. 5 has shown that detecting the alternative Nucleotide of let-7a miRNA designs, and the target-specific zone of its middle probe and INVADER oligonucleotide all is that 11 Nucleotide are long.It all is linear design that SEQ ID NO:50-51 provides described INVADER and probe.SEQ ID NO:6 contains the probe oligonucleotides that has formed loop-stem structure; SEQ ID NO:5, the INVADER oligonucleotide of formation loop-stem structure); SEQ ID NO:5-6, two probe and INVADER oligonucleotide that the stem ring is all arranged.

B.10 aggressiveness probe and 12 aggressiveness INVADER oligonucleotide design

Fig. 5 has shown the alternative oligonucleotide design of a cover that detects let-7a miRNA, and the target-specific zone of wherein said probe comprises 10 Nucleotide, and those target-specific zones of INVADER oligonucleotide comprise 12 Nucleotide.It all is linear design that SEQ ID NO:52-53 provides INVADER and probe oligonucleotides.SEQ ID NO:2 contains the probe oligonucleotides that has formed loop-stem structure; SEQ ID NO:1 has formed the INVADER oligonucleotide of loop-stem structure.

2.Let-7a the temperature optimization spectrum (profile) of the alternative oligonucleotide design of the detection of miRNA

The temperature optimization experiment is carried out as follows.The summation mixture is made into to be used for 24 reactions.Each reaction contains following:

Stock concentration	The volume of each reaction	Final concentration
			The 2.5X primary reaction damping fluid of CLEAVASE XII enzyme (as describing among the embodiment 5)	4μl	1X
10 μ M probes *	1μl	1μM
			100 μ M INVADER oligonucleotide *	1μl	10μM
60ng/μl CLEAVASE 12	0.5μl	30ng
			Water	2.5μl	N/A
30pM miRNA (for the temperature optimization of 11 aggressiveness) or 10nM miRNA (for the temperature optimization of 10 aggressiveness probes/12 aggressiveness INVADER oligonucleotide)	1μl	3pM or 1nM
			20ng/ μ l tRNA (for no target contrast)	1μl	2ng
Summation	10μl

^*The multiple combination of probe and INVADER oligonucleotide is used for this experiment shown in Figure 16-17.

Describe among the embodiment 3 of secondary reaction mixture such as let-7.In the time of suitable, make the ARRESTOR sequence, and extend 6 bases towards described probe 5 ' end with complete ring and the target-specific zone of corresponding (compliment) described probe.

Under the situation of 11 aggressiveness temperature optimization experiments, primary reaction carried out 1 hour at 50 ± 9 ℃, be afterwards as describe among the embodiment 2 60 ℃ of following secondary reactions of 15 minutes.For described 10 aggressiveness probes and 12 aggressiveness INVADER oligonucleotide together, primary reaction carried out 15 minutes at 50 ± 9 ℃, was 15 minutes secondary reaction at 60 ℃ afterwards.

Wherein the target-specific of INVADER and probe oligonucleotides partly is that the result of the design of 11 Nucleotide length is presented among Figure 18.Figure 18 A has shown the temperature optimization spectrum of each design.Figure 18 B has shown the execution to greatest extent of each standardization, comprises the Optimal Temperature of each design.Wherein the target-specific of probe oligonucleotides partly is 10 bases, and the INVADER oligonucleotide is that the result of the design of 12 bases is presented among Figure 19.Figure 19 A has shown the temperature optimization spectrum, and Figure 19 B has shown the execution to greatest extent of each standardization.

Relative stability different and different of the miRNA-oligonucleotide hybridization body of reaction conditions and formation are depended in the design that these results' inspection prompting, design cause carrying out to greatest extent.For example, when the target-specific zone of two oligonucleotide all was 11 bases length, described probe target specific regions had 49 ℃ expection Tm, and there is 37 ℃ expection Tm in described INVADER target-specific zone.Under this situation, the interactional stabilization of INVADER oligonucleotide-miRNA provides the test that improves to implement to this design.Yet, be that 10 aggressiveness and INVADER oligonucleotide are the let-7a designs of 12 aggressiveness for probe, the target-specific zone of described two oligonucleotide has the Tm that approximately equates.Under this situation, two oligonucleotide all are that cyclic design and operation ground is best.

3. use the LOD of the let-7a of two optional designs

Experiment as be provided with describing among the embodiment 3 with as described in the dicyclo design and as described in the LOD that designs of monocycle, wherein the INVADER oligonucleotide has formed loop-stem structure.

Measuring the reaction of LOD carries out with four duplicate samples.Reaction mixture contains following reagent (final concentration):

Stock concentration	The volume of each reaction	Final concentration
			The 2.5X primary reaction damping fluid of CLEAVASE XII enzyme	4μl	1X
10 μ M probes/200 μ M INVADER oligonucleotide mixtures (as the sequence of pointing out among Figure 20)	0.5μl	1 μ M probe/20 μ M INVADER oligonucleotide
			60ng/ μ l CLEAVASE XII enzyme	0.5μl	30ng
Summation	5μl

The aliquot of 5 μ l miRNA joins in the hole of containing reaction mixture with the final concentration of pointing out among Figure 20.The optimized temperature of each design that primary reaction is measured in embodiment 8B (the INVADER oligonucleotide of Cheng Huan is designed to 50 ℃, and dicyclo is designed to 53 ℃) was carried out 1.5 hours.Secondary reaction is as being provided with in embodiment 2 and 3 with describing and carrying out 1 hour at 60 ℃.

Result among Figure 20 has shown the net signal that produces, and it is the function of miRNA molar weight.The linearity range of described chart is pointed out to use described INVADER loop design to design from the miRNA of specified quantitative than dicyclo and is produced more signal.Similarly, the inspection of showing described in Figure 20 shows: be higher than the bigger of zero multiple numeric ratio monocycle design in each miRNA level.Two designs (are equal to 2.68 * 10 of about 16,000 molecules at the minimum concentration of test ^-20Mole or 26.8 narrow moles (zeptomole)) obtained enough FOZ.

4. the ARRESTOR oligonucleotide for brachymemma of total length

Experimentize with evaluation Example as the relative performance of the total length ARRESTOR molecule as shown in Fig. 4 and 12 for the ARRESTOR molecule that blocks, the ARRESTOR molecule of wherein said total length its 5 ' terminal extension around ring, the total length of penetration probe miRNA specific regions and to 5 ' flap zone, the ARRESTOR molecule of described brachymemma only is complementary to the miRNA specific regions of probe and the part of described 5 ' flap, but does not extend to annular section or farther zone.Reacting following is provided with to detect synthetic let-7amiRNA:

Component	Stock concentration	The amount that each reaction adds
			PI mixture (probe SEQ ID NO:6; INVADER oligonucleotide SEQ ID NO:5)	10 μ M probes, 50 μ M INVADER oligonucleotide	1μl
CLEAVASE XII enzyme	(60ng/μl)	0.5μl
			Water
The primary reaction damping fluid	2.5X	4μl
			Summation		6μl

The aliquot of 6 μ l primary reaction mixtures joins in the dull and stereotyped suitable hole of microtitration, adds the aliquot of the synthetic let-7a miRNA of 4 μ l that goes out as the following table middle finger or the aliquot of the 10ng/ μ l tRNA that 4 μ l are dissolved in distilled water afterwards.Elementary INVADER is reflected at 53 ℃ and hatched 1.5 hours.

The following adding of the aliquot of secondary reaction mixture:

The ARRESTOR of total length
			Component	Stock concentration	The amount that adds
The ARRESTOR of ARRESTOR total length, SEQ ID NO:7; The ARRESTOR that blocks, SEQ ID NO:54	40μM	1μl
			MOS SRT(SEQ ID NO：22)	1.5μM	1μl
FRET FAM(SEQ ID NO：21)	10μM	1μl
			Water		2μl

Secondary reaction was hatched 1.5 hours at 60 ℃.Microtitration is dull and stereotyped carries out reading as embodiment 2 with describing.The result is presented among Figure 17.

These results show, when ARRESTOR oligonucleotide total length or that block that is complementary to described probe miRNA specificity part was used for secondary INVADER reaction, the generation of signal or detectability were not different significantly.

B. use linear probe and INVADER oligonucleotide optionally to design

Optional design is tested, and wherein said probe and INVADER oligonucleotide all contain universal sequence, and do not have oligonucleotide to form hair clip.The synoptic diagram of described design is presented among Fig. 4.Described universal sequence is present in 3 ' end of 5 of INVADER oligonucleotide ' end and probe oligonucleotides.Add short complementary and " catch " oligonucleotide, and comprise 2 '-oxygen-methyl residue, allow it in the presence of miRNA (for example SEQ ID NO:60), to promote coaxial accumulation.Produce the design of miR-15 (SEQ ID NO:58,59 and 60) and mir-135 (SEQID NO:63-65).Though initial design has caused high non-specific background signal in the presence of no miRNA target, pointed out that it is feasible detecting miRNA with so general capture oligo.

Embodiment 9

Nucleotide residue 2 in the ring '-oxygen-methylated effect

This embodiment described be intended to evaluate be used for detecting described probe of being integrated in of miRNA and INVADER oligonucleotide some or all 2 '-oxygen-methyl residue by 2 '-experiment of the effect that the deoxidation residue replaces.All designs that present in the foregoing embodiments all comprised as 2 in the ring zone as described in describing among the embodiment 2 '-oxygen-methyl residue.Experimentize with test in the INVADER that detects let-7a miRNA design and the probe oligonucleotides some or all 2 '-effect that oxygen-methyl residue is replaced by 2 ' deoxidation residue.

Fig. 5 has shown the let-7a design of improvement.SEQ ID NO:5-6 contain as describe among the embodiment 22 '-oxygen-methyl residue.Design among the SEQ ID NO:73-74 all contains 2 ' deoxidation residue in all positions; And those designs in SEQ ID NO:75-76, in the described stem part of contiguous target, contain 2 '-oxygen-methyl residue.

Carry out the INVADER reaction with the signal generation of three different designs of comparison and the optimization of temperature.Reaction comprises 100pM synthetic miRNA, 1 μ M probe and 10 μ M INVADER oligonucleotide as describing in the LOD test among the embodiment 8.Primary reaction shown in temperature carried out 15 minutes; Secondary reaction was carried out 5 minutes at 60 ℃.

The result of INVADER test is presented among Figure 21, show wherein said loop-stem structure comprise 2 '-design of oxygen-methyl residue produces maximum signals, be afterwards the base of wherein contiguous target comprise 2 '-design of oxygen-methyl residue.Comprise 2 fully '-oligonucleotide of deoxidation residue produces the signal of minimum level.

The following design of another set of experiment is to test extra design variation: the more probe and the INVADER oligonucleotide of bob folder are arranged, probe and INVADER oligonucleotide more stable ring or that shorter ring is optionally arranged are arranged, have only three 2 '-probe and the INVADER oligonucleotide of oxygen-methyl residue.The following setting of primary reaction is with the detection of test miR-15.

To testing following probe/uniting of INVADER oligonucleotide.

Probe		The INVADER oligonucleotide
				1544-71-01	SEQ ID NO：55	1544-71-02	SEQ ID NO：56
1544-71-01	SEQ ID NO：55	1796-43-02	SEQ ID NO：68
				1544-71-01	SEQ ID NO：55	1796-43-04	SEQ ID NO：70
1544-71-01	SEQ ID NO：55	1796-43-06	SEQ ID NO：56
				1796-43-01	SEQ ID NO：67	1544-71-02	SEQ ID NO：56
1796-43-03	SEQ ID NO：69	1544-71-02	SEQ ID NO：56
				1796-43-05	SEQ ID NO：71	1544-71-02	SEQ ID NO：70
1796-43-03	SEQ ID NO：69	1796-43-04

The primary reaction mixture is following to be made:

The primary reaction component	Stock concentration	The amount that adds
			Probe oligonucleotides (as above table is pointed out)	40μM	0.25μl
The INVADER oligonucleotide	40μM	0.25μl
			CLEAVASE XII enzyme	60ng/μl	0.5μl
The primary reaction damping fluid	2.5x	4μl
			Summation		5μl

The aliquot of 5 μ l primary reaction mixtures joins 5 μ l synthetic miR-15RNA:0,0.1 Ah's mole (amole), 0.33 Ah's mole, 1.09 Ah's moles with following amount at end.Primary reaction was hatched 2 hours at 52.5 ℃.

The secondary reaction mixture is following to be made:

The secondary reaction component	Concentration	The amount that adds
			FAM FRET oligonucleotide (SEQ ID NO:21) and secondary reaction target (SRT) (SEQ ID NO:40)	13.4μl FAM FRET 2μM SRT	0.75μl
ARRESTOR oligonucleotide (SEQ ID NO:66)	54μM	0.75μl
			Water		3.5μl
Summation		5μl

The aliquot that adds 5 μ l is reflected at 600 ℃ and hatched 45 minutes.

As a result, with relative fluorescence unit (RFU), present as follows.

Probe	1544-71-01						1544-71-01
												INVADER	1544-7l-02			Only	FOZ		1796-43-02			Only	FOZ
1.09	655	658	662	566	7.13	1.09	706	738	777	636	7.12
												Ah's mole						Ah's mole
0.33	314	262	256	185	3.00	0.33	281	287	290	182	2.75
												Ah's mole						Ah's mole
0.10	122	138	134	39	1.42	0.10	149	150	153	47	1.45
												Ah's mole						Ah's mole
0	88	93	96			0	104	101	107
												Ah's mole						Ah's mole

Probe	1796-43-01						1796-43-03
												INVADER	1544-71-02			Only	FOZ		1544-71-02			Only	FOZ
1.09	1689	1744	1895	146	1.09	1.09	882	869	847	702	5.27
												Ah's mole						Ah's mole
0.33	1655	1717	1817	99	1.06	0.33	335	341	34l	175	2.06
												Ah's mole						Ah's mole
0.10	1692	1693	1695	63	1.04	0.10	196	209	196	36	1.22
												Ah's mole						Ah's mole
0	1636	1601	1654			0	169	165	159
												Ah's mole						Ah's mole

	1544-71-01						1544-71-01
													1796-43-04			Only	FOZ		1796-43-06			Only	FOZ
1.09	676	688	693	579	6.43	1.09	625	562	579	501	6.69
												Ah's mole						Ah's mole
0.33	274	275	264	164	2.54	0.33	229	215	204	128	2.45
												Ah's mole						Ah's mole
0.10	153	137	143	38	1.35	0.10	126	121	112	32	1.36
												Ah's mole						Ah's mole
0	111	107	102			0	94	87	83
												Ah's mole						Ah's mole

	1796-43-05						1796-43-05
													1544-71-02			Only	FOZ		1796-43-06			Only	FOZ
1.09	806	824	773	708	8.64	1.09	772	752	704	631	6.65
												Ah's mole						Ah's mole
0.33	280	280	262	181	2.96	0.33	260	252	25l	143	2.28
												Ah's mole						Ah's mole
0.10	144	145	139	50	1.54	0.10	140	142	139	29	1.26
												Ah's mole						Ah's mole
0	91	95	92			0	115	109	111
												Ah's mole						Ah's mole

These results suggest, wherein probe oligonucleotides contain the hair clip of brachymemma and comprise 2 '-design of the design of the high stability Fourth Ring probe oligonucleotides of oxygen-methyl residue and initial INVADER oligonucleotide (2 '-oxygen-methyl residue, the TTTT ring, the long hair folder) combined, can produce higher a little FOZ value.Otherwise the optional design oligonucleotides cover of none provides any raising on initial design.Noteworthy is that all DNA oligonucleotide provide those FOZ values that approximately are equal to chimeric probe and the acquisition of INVADER oligonucleotide with the combination of chimeric probe oligonucleotide at first.In some applications, the replacement of all DNAINVADER oligonucleotide is expected, synthesize cost to reduce oligonucleotide, and preparation can not sacrificed detectability.

Experimental results show that further the generation of gathering compensation subcell optimization (suboptimal) signal with special oligonucleotide by the more RNA of adding (for example total RNA of lysate, purifying, synthetic miRNA) in reaction is possible.Similarly, pointed out by the extra experiment of gel-purified that as the various oligonucleotide of describing among the embodiment 1 (being probe, INVADER, ARRESTOR or its multiple combination) the standard gel purifying of all three types of oligonucleotide has provided maximum signal.If other oligonucleotide, i.e. INVADER and ARRESTOR oligonucleotide desalination after synthetic, the signal level that obtains so to approximate greatly with the maximum horizontal of gel-purified probe is possible.

Embodiment 10

Detection from miRNA expression among total RNA of multiple types of organization

This embodiment has described the experiment of carrying out for the appropriateness of test I NVADER test, with Detection and Extraction different miRNA type in total RNA of histological types.For the evaluation of tissue expression of specific gene, the temperature optimization of each design and LOD are earlier determined.

1.INVADER and probe oligonucleotides design

Design INVADER pilot oligonucleotide is to detect miR-15, miR-16 and miR125bmiRNA kind.The design of these tests is presented among Fig. 5.The design of Let-7a and miR-135 has description respectively in embodiment 2 and 6.

2. the mensuration of temperature optimization and LOD

For each these oligonucleotide combination, the temperature optimization experiment is implemented as describing ground among the embodiment 8.Each primary reaction comprises 1nM target miRNA, and carries out in 50 ± 9 ℃ temperature range 15 minutes.Secondary reaction is as describing among the embodiment 2, and carries out 1 to 1.5 hour at 60 ℃.Optimal Temperature is as follows:

let-7a	53℃
		miR-15	53℃
miR-16	56℃
		miR-125b	52℃
miR-135	45℃

In case the acquisition temperature optimization is measured among the LOD of each miRNA kind such as the embodiment 8 with describing.All LOD are 30 narrow moles.

3. gene expression profile (profiling)

Gene expression profile carries out on total RNA of 20 histological types in extraction.Total RNA buys (Palo Alto, CA, catalog number (Cat.No.) K4008-1, Human TotalRNA Master Paneln) from Clontech.For let-7a, the total RNA to 50ng in each reaction tests; For other miRNA kind, total RNA of 100ng is tested.Institute responds and is provided with as described in example 8 above; Primary reaction carried out 1.5 hours in Optimal Temperature; Secondary reaction is as described above.

The gene expression profile of each miRNA kind is presented among Figure 23.These results point out that the INVADER test can be used to check the expression of miRNA in the histological types.These data further point out let-7a and miR-125b to be expressed in the various tissues; As if other miRNA kind more have specificity for the types of organization of limited quantity.

Embodiment 11

Different oligonucleotide length is to the effect of the INVADER test detection of miRNA

This embodiment has described the influence of the change of probe and INVADER oligonucleotide length to the miRNA detection of 22 Nucleotide of let-7a.In particular, the detection of following two kinds of miRNA has been compared in these experiments: form the detection of the desirable interactional miRNA of accumulation between the end of described probe and INVADER oligonucleotide and form 5 ' and the detection of 3 ' eclipsed miRNA and described 5 ' and 3 ' end obtain the detection of the miRNA of single Nucleotide breach.

Figure 24 has shown the result who analyzes three types of designs.SEQ ID NO:5-6 has shown between the lateral end of the probe of 22 Nucleotide targets and Cheng Huan and INVADER oligonucleotide and has perfectly piled up.In SEQ ID NO:83-84, probe and INVADER oligonucleotide have all extended a base, obtained 5 ' and 3 ' overlapping.In SEQ ID NO:85-86, probe and INVADER oligonucleotide with respect to the design of SEQ ID NO:5-6 all brachymemma a base, obtained a Nucleotide breach at two ends.

The INVADER test is set to test the execution that these oligonucleotide combinations detect for synthetic let-7amiRNA.Reaction is carried out as described in example 8 above, comprises 100pM synthetic let-7a miRNA, 1 μ M probe and 10pM INVADER oligonucleotide.Primary reaction carried out 15 minutes at 53 ℃; Carried out 5 minutes at 60 ℃ among secondary reaction such as the embodiment 2 with describing.The result is presented among Figure 24.

These data point out in this experiment, reduce approximately 30% in the overlapping generation that causes signal of the single Nucleotide of two ends of miRNA target, and Optimal Temperature reduces by 2 ℃.Yet a Nucleotide breach of two ends of described target does not reduce the generation of signal, but it has reduced by 5 ℃ of peak optimization reaction temperature really.

Embodiment 12

MiRNA and precursor RNA and with the difference of coding DNA

Whether can distinguish miRNA target itself and experimentize in order to measure INVADER miRNA test from the precursor RNA of miRNA target and from the DNA of the described miRNA that encodes.

A. precursor cross reaction property testing

External the transcribing of precursor let-7 RNA (SEQ ID NO:87) analyzes to measure it whether contain any fragment of imitation let-7a miRNA by capillary electrophoresis.It is about 45 Nucleotide that the shortest pollution fragment is estimated.The LOD reaction basically as carrying out under the precursor that goes out as the following table middle finger or synthetic 5 ' P let-7a miRNA concentration among the embodiment 3 with describing.The PI mixture contains 10pM probe SEQ ID NO:6 and 100 μ M INVADER oligonucleotide SEQ ID NO:5.Primary reaction carried out 1 hour at 53 ℃; Basically carried out 1 hour at 58 ℃ as the secondary reaction of the secondary reaction mixture (FRET probe SEQ ID NO:21, SRT SEQ ID NO:22 and ARRESTOR SEQ ID NO:7) of description among the embodiment 3.This result of experiment points out that this miRNA test has about 4% cross reactivity for precursor RNA.

B.RNA is to the difference of DNA signal

For the let-7a miRNA that detects in the cell lysate reacts as described in example 7 above.Before the detection with the INVADER test, the aliquot of 1 μ l, 8 μ g/ μ l RNAse A (Qiagen company) joins in the 80 μ l cell lysates and at 37 ℃ hatched 2.25 hours.The sample that RNAse A handles can not produce the signal on any background, points out to lack the signal that produces in the test of RNAse A and results from miRNA target rather than the detection of coding DNA (Figure 16).Further test, wherein RNAse A is adding before the primary reaction or before the secondary reaction.When RNAse A added fashionablely before primary reaction, there is not signal to produce, consistent with former result.When RNAse A adds after primary reaction, do not observe the signal of losing, point out that further the signal that detects is because RNA and RNAse do not have disadvantageous effect to other reactive component of for example CLEAVASE enzyme.

Embodiment 13

The detection of dual form miRNA

Generation is for the oligonucleotide design of miR-124a.These oligonucleotide can be used to detect the miRNA of two natural generations, long 21 Nucleotide, another 22 Nucleotide.

The temperature optimization reaction is used 1nM synthetic miRNA target, 25 minutes primary reaction and 15 minutes secondary reaction basically as being set up among the embodiment 3 with describing.The oligonucleotide that uses in these reactions is listed in (SEQ ID NO:90-92) among Fig. 5.The TEMPERATURE SPECTROSCOPY (profile) of the miRNA target of two different lengthss is presented among Figure 22 and points out that identical oligonucleotide design can be used to detect two targets.

Embodiment 14

The oligonucleotide design that siRNA detects

Can similarly be used to detect siRNA with those similar methods of describing among the aforesaid embodiment.Figure 25 has set forth two optional INVADER test design that beta-actin siRNA detects.This siRNA is at Harborth, people such as J., and cell science magazine (Journalof Cell Science), 114:4557-4565 has description in (2001).There are justice and antisense strand to have a design for each; Detect the oligonucleotide of the exemplary embodiment of this siRNA and list among Fig. 6, SEQ ID NO:101-106.

All publications and the patent mentioned in describing in detail are above quoted as a reference herein.The various modifications and the variation of the method and system that the present invention describes it will be apparent to those of skill in the art, and do not depart from the field of the invention and spirit.Although the present invention has description together with special preferred embodiment, be to be understood that desired the present invention should be limited to so special embodiment inadequately.Certainly, be conspicuously to be intended to fall among the following claim scope for the various modifications of carrying out pattern described in the invention to molecular biology, gene or those skilled in the relevant art.

Claims (31)

1. method comprises:

A) the RNA interfering target is hybridized at least a nucleic acid, described nucleic acid contains and is not complementary to described RNA interfering target to produce the sequence of detection architecture; And

B) detect described detection architecture.

2. according to the process of claim 1 wherein that described RNA interfering target is miRNA.

3. according to the process of claim 1 wherein that described RNA interfering target is siRNA.

4. according to the method for claim 3, wherein said siRNA is double-stranded.

5. according to the process of claim 1 wherein that described detection architecture comprises the invasive cutting structure.

6. according to the method for claim 2, wherein said detection architecture comprises first and second oligonucleotide that dispose for the invasive cutting structure that forms the described miRNA of associating.

7. according to the method for claim 2, wherein said detection architecture comprises first oligonucleotide that disposes for the invasive cutting structure that forms the described miRNA of associating.

8. according to the method for claim 6, wherein said first oligonucleotide comprises 5 ' part and 3 ' part, and wherein said 3 ' part disposes in order to hybridize in described target sequence, and wherein said 5 ' part disposes in order not hybridize in described target sequence.

9. according to the method for claim 6, wherein said second oligonucleotide comprises 5 ' part and 3 ' part, and wherein said 5 ' part disposes in order to hybridize in described target sequence, and wherein said 3 ' part disposes in order not hybridize in described target sequence.

10. according to the process of claim 1 wherein that described detection comprises the use of invasive cutting test.

11. according to the process of claim 1 wherein that described detection architecture comprises the annular oligonucleotide of hybridizing in described miRNA to produce annular detection architecture.

12. according to the method for claim 11, wherein said detection comprises the use of rolling-circle replication test.

13. according to the process of claim 1 wherein that described detection comprises the use that is selected from following detection test: order-checking test, polymerase chain reaction test, cross experiment, employing are complementary to cross experiment, microarray test, the test of pearl array, primer extension test, enzyme mispairing cutting test, side chain cross experiment, NASBA test, molecular beacon test, circle probe test, ligase chain reaction (LCR) test, the test of invasive cutting structure, ARMS test and the sandwich hybridization test of the probe of sudden change.

14. according to the process of claim 1 wherein that described detection carries out in cell lysate.

15. detect multiple different miRNA according to the process of claim 1 wherein.

16. according to the method for claim 15, wherein said multiple miRNA comprises a miRNA and the 2nd miRNA, described the 2nd miRNA is the described miRNA with polymorphism.

17., comprise further and detect second nucleic acid target according to the method for claim 1.

18. according to the method for claim 17, wherein said second nucleic acid target is RNA.

19. according to the method for claim 18, wherein said second nucleic acid target is selected from U6 and GAPDH.

20. according to the method for claim 2, wherein said miRNA is selected from Let-7, miR-1, miR-135, miR-15, miR-16, miR125b, miR-1d and miR124a.

21. a test kit, it comprises the nucleic acid that disposes in order to form detection architecture when hybridizing in the RNA interfering target sequence, and wherein said nucleic acid comprises the sequence that is not complementary to described RNA interfering target sequence.

22. according to the test kit of claim 21, wherein said detection architecture comprises the invasive cutting structure.

23. according to the test kit of claim 22, wherein said first oligonucleotide comprises 5 ' part and 3 ' part, wherein said 3 ' part disposes in order to hybridize in described target sequence, and wherein said 5 ' part disposes in order not hybridize in described target sequence.

24. according to the test kit of claim 22, wherein said second oligonucleotide comprises 5 ' part and 3 ' part, wherein said 5 ' part disposes in order to hybridize in described target sequence, and wherein said 3 ' part disposes in order not hybridize in described target sequence.

25. according to the test kit of claim 21, wherein said detection architecture comprises the annular oligonucleotide of hybridizing in described miRNA to produce annular detection architecture.

26. according to the test kit of claim 25, wherein said detection architecture is the detection architecture that is used for the rolling-circle replication test.

27. according to the test kit of claim 21, wherein said RNA interfering target is miRNA.

28. according to the test kit of claim 21, wherein said RNA interfering target is siRNA.

29. according to the test kit of claim 27, wherein said miRNA is selected from Let-7, miR-1, miR-135, miR-15, miR-16, miR-lb, miR-124a and miR125b.

30. according to the test kit of claim 21, wherein said test kit for detect miRNA target and at least one other the RNA target and dispose.

31. according to the test kit of claim 21, wherein said test kit disposes in order to detect the target sequence of RNA interfering described in the cell lysate.

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