CN1103822C - Method for detecting target nucleic acid - Google Patents

Abstract

The present invention relates to a method for detecting target nucleic acid. The present invention comprises the following steps that (i) amplification products are obtained through the amplification of target nucleic acid, a kind of polymerase, a first primer and a second primer are used during amplification, the first primer has or does not have fragments which are not adjacent to a first guide sequence, the second primer has or does not have fragments which are not adjacent to a second guide sequence, amplification is carried out in the existence of oligonucleotide which can not be used as a primer of the polymerase, and the oligonucleotide has five consecutive nucleotides which are completely complementary with the at least five consecutive nucleotides of the first primer; (ii) the amplification of the target nucleic acid is monitored so as to detect the target nucleic acid. Special attention is paid to the drawing in the specification, which illustrates the method, and the primers in the drawing contain the fragments which are not adjacent to primer sequences.

Description

Detect the method for target nucleic acid

The recent development of polymerase chain reaction (PCR) provides important instrument (Mullis.K.B etc., United States Patent (USP) the 4th, 683, No. 195 and the 4th, 683, No. 202) for detecting the low concentration nucleic acid sequence.In PCR, extend primers by two oligonucleotide and determine its terminal target sequence to be amplified, so that provide more template for further amplified reaction by multiple enzymatic reaction circulation.Therefore, a small amount of target sequence can obtain amplification with exponential manner, and is easy to detect.

The major defect of PCR is very easily to produce by product, and this causes by non-specific guiding, for example, and the nucleic acid-templated self-priming that guides and/or extend primer at random.Therefore, when the target sequence that need exist with low concentration with the amplification of more amplification cycles number of times, the product of non-specific guiding can obviously reduce the susceptibility of PCR.

The defective that another of PCR is relevant therewith will have a separating step before being to detect amplified production.According to Standard PC R condition, the prerequisite that detects the amplified target sequence is to isolate the target sequence of amplification from non-specific guiding product.An obstacle that makes the PCR operation automation is to carry out the homology amplified reaction, and so-called homology amplified reaction increases in same reaction vessel exactly and detects.In addition, need separate that the PCR mixture is polluted easily in separating step, contamination of heavy has seriously limited the potential application of PCR in the routine clinical diagnosis.

Existing bibliographical information, the someone attempts to develop and a kind of homogeneous phase detection method, is used for amplification and detects.Holland etc. (1991.Proc.Natl.Acad.Sci., USA 88:7276) have similar report, and they utilize 5 of Taq polysaccharase ' → 3 ' exonuclease activity, follow pcr amplification to produce detection signal, thereby detect.Yet, also need carry out a separating step subsequently to detect the signal that exonuclease produces.Higuchi etc. (1992 Bio/Technology 10:413) reported a kind of homogeneous phase detection method, this method be based on ethidium bromide inserted double-stranded amplified production after enhanced fluorescence monitor.This detection method does not have specificity to target sequence, thereby is vulnerable to the interference of existing any double-stranded DNA.Another kind method is to utilize fluorescence polarization to measure the hybridization of fluorescent core acid probe or fluorescent primer extension products (German, A.J. etc., No. the 382nd, 433, EUROPEAN PATENT OFFICE's publication number).Probe and unreacted probe that this detection method can be distinguished hybridization or extend, the former has higher molecular weight (thereby the fluorescence polarization value reduces).Yet the existence with not hybridization probe of high polarization value influences the polarization value that observes consumingly, thereby background is higher.

Two fluorescence part extremely near the time radiationless energy shift and can be used as a kind of effective signal detecting mode.Based on the existing bibliographical information (Patel etc. of the homogeneous immunoassay of fluorescence energy transfer.No. the 137th, 515, european patent application).Fluorescence energy transfer also is used to measure nucleic acid hybridization (Heller etc., No. the 4th, 996,143, No. the 070th, 685, european patent application and United States Patent (USP)).In these applications, when the probe that has different fluorescence part since with a target nucleic acid hybridization mutually extremely near the time, just produced the fluorescence energy transfer signal.

On the one hand, the present invention relates to a kind of method that detects target nucleic acid.This method may further comprise the steps: (i) amplifying target nucleic acid obtains amplified production, use polysaccharase during amplification, first primer and second primer, first primer has or does not have and the non-conterminous fragment of first homing sequence, second primer has or does not have and the non-conterminous fragment of second homing sequence, amplification is carried out in the presence of a kind of oligonucleotide, this oligonucleotide can not be as the primer of described polysaccharase, wherein, this oligonucleotide has at least 5 (preferably at least 8) successive Nucleotide that at least 5 (preferably at least 8) successive Nucleotide are complementary to first primer fully; (ii) detect the existence of target nucleic acid by the monitoring amplification procedure.

The primer that first and second primers are equivalent in the PCR method is right, and they are being aggregated before enzyme extends, respectively with target nucleic acid in the hybridization of first and second homing sequences.Among the application, " polysaccharase " refers to both have the nucleotide polymerase activity, can remove the catalyzer of " downstream " single stranded oligonucleotide again.

Above-mentioned oligonucleotide is " sealing oligonucleotide " among the present invention.For simplicity, as long as any oligonucleotide meets following condition, all be called " sealing oligonucleotide " in this application: (a) can not be as the PCR primer, (b) have at least 5 continuous nucleotides that at least 5 successive Nucleotide are complementary to a primer (have or fragment that none is adjacent) fully.Sealing oligonucleotide sealing primer is meant it and primer hybridization, and making in primer and the target nucleic acid accordingly, homing sequence bonded probability reduces.In a preferred embodiment of this method, amplification step is to exist at the same time under the condition of the another kind of primer sealing oligonucleotide that is used for second primer to carry out.

5 base fully-complementary sequences can form between adjacent segment of sealing oligonucleotide and primer or non-conterminous fragment.The adjacent segment of primer be with target nucleic acid in homing sequence complementary sequence.Otherwise, the non-conterminous fragment of primer for not with target nucleic acid in homing sequence complementary sequence.

The preferred length of primer of the present invention and sealing oligonucleotide is 10-50 Nucleotide (more preferably 15-40 Nucleotide).The working concentration of sealing oligonucleotide is subjected to the influence of several factors, alters a great deal, and these factors comprise the complementary degree, test temperature of sealing oligonucleotide and its corresponding primer etc.Generally speaking, those skilled in the art need not too much test and can determine this working concentration.The mol ratio of sealing oligonucleotide and its corresponding primer acceptable normally between 0.3-5.0 (or 0.5-2.5).

Detecting step is to be undertaken by the target nucleic acid amplified production on monitoring gel behind the electrophoresis.Perhaps two kinds of fluorophores can be covalently attached to respectively first or (second) primer and corresponding sealing oligonucleotide thereof on, in two kinds of fluorophores, a kind of is the donor fluorophore, another kind is an acceptor fluorescence group.When first primer and its sealing oligonucleotide hybridization, donor fluorophore and acceptor fluorescence group are extremely approaching, resonance energy can take place each other shift.After sealing oligonucleotide primer corresponding with it is like this with the fluorophore mark, the fluorescent emission that just can monitor acceptor fluorescence group with exciting light radiation donor fluorophore the time changes, the amplification of target nucleic acid is carried out quantitatively, because this variation has reflected the primer that is closed and sealed the degree that oligonucleotide unwinds and mixes the target nucleic acid amplified production subsequently.

On the other hand, the present invention relates to the test kit that is used to detect target nucleic acid.For example, a kind of detection kit of the present invention can comprise: (i) a pair of primer (10-50 base or 15-40 base), and they are used to amplifying target nucleic acid with polysaccharase, and primer has or none and the non-conterminous fragment of its primer sequence; (ii) seal oligonucleotide (10-50 base or 15-40 base), it can seal in two primers one.The corresponding primer with it of sealing oligonucleotide is preferably all used the fluorophore mark in the above described manner.Sealing oligonucleotide and corresponding primer thereof can provide by doublet (be " the special detection doublet " of fluorophore mark, see in the middle of Fig. 1 and Fig. 2 top, discuss and see below).This test kit preferably also comprises one of following reagent or whole: the another kind sealing oligonucleotide of a kind of polysaccharase and other primers of sealing.

In another embodiment, a kind of test kit of the present invention can comprise: (i) oligonucleotide of first fluorophore mark (10-50 base or 15-40 base), and it can not be as the primer of polymeric enzymatic amplification; The (ii) oligonucleotide of second fluorophore mark (5-30 base or 7-15 base), it has free 3 ' OH.Two kinds of oligonucleotide by at least 5 complete complementary oligonucleotide ligands of (preferably at least 8) successive to and the phase mutual cross, first oligonucleotide stretches out second oligonucleotide 3 ' terminal 1-12 base (preferably 4-8 base).And in first and second fluorophores, a kind of is the donor fluorophore, and another kind is an acceptor fluorescence group, and they are extremely approaching after hybridization, resonance energy can take place each other shift.First and second oligonucleotide can provide by doublet, i.e. " general detection doublet " (seeing following discussion).The preferred reagent box also comprises other reagent, as ligase enzyme, kinases with have or do not have the polysaccharase of 5 ' → 3 ' exonuclease activity, ligase enzyme and the kinases polysaccharase of 5 ' → 3 ' exonuclease activity (or have or do not have) can be used to any selected primer is connected on the general detection doublet, thereby the latter is transformed into special detection doublet.

Above-mentioned special and general detection doublet, and the invalid oligonucleotide of forming one of the doublet chain all belongs to scope of the present invention.

Other features and advantages of the present invention can be found out from the description of the following drawings and preferred embodiment and claims.

At first introduce accompanying drawing.

Fig. 1 is a kind of embodiment of method of the present invention.

Fig. 2 is the another embodiment of method of the present invention.

Fig. 3 is a gel photograph, shows that the sealing oligonucleotide reduces the effect of non-specific guiding.

Fig. 4 is a column diagram, shows the sealing oligonucleotide monitoring specificity guiding of using the fluorophore mark.

Fig. 5 is a graphic representation, shows resolving power and the susceptibility of using two kinds of different target sequences of two kinds of probe in detecting of the present invention.

" amplification " is that general reference uses polymerase and the one pair of primer to produce the mistake of any specific nucleic acid sequence herein Journey, specific nucleic acid sequence i.e. " target nucleic acid sequence " or " target nucleic acid ", and the amount that generation more initially exists is wanted Many.

The present invention relates to a kind of method, the method is mixed the amplified production of specific nucleic acid by monitoring primer Process, detect the existence of specific nucleic acid sequence. Among the present invention, the sealing oligonucleotides is used to prevent Non-specific guiding takes place in the amplification procedure. In a preferred embodiment, few by the fluorescence monitoring sealing Nucleotides and unwinding of its complementary primer and detect the target sequence of amplification.

Amplification depends on each to the hybridization of primer and its particular pilot sequence, and the particular pilot sequence refers to initial One section sequence in nucleic acid-templated, the complementary degree of it and primer is the highest, and the easiest being hybrid with it. Remove Outside the particular pilot sequence, the initial nucleic acid template can also comprise non-specific guiding sequence, primer and its Also can hybridize. Non-specific guiding is that one section sequence outside the specific nucleic acid sequence of instigating in the template obtains The phenomenon of amplification, it comprise such as the reaction of the sequence hybridization outside the primer sequence in primer and the template with And such as since two primers that the intermolecular interaction of primer causes self guide (Chou etc. Nucleic Acid Research, (1992) 20:1717).

Primer is one section oligonucleotides, can be obtained or by the preparation of organic method by restriction enzyme digestion product purifying Obtain, under suitable condition, it can be used as the synthetic starting point of primer extension product that is complementary to target sequence. Primer of the present invention is except comprising the sequence with the particular pilot sequence complementation of initial nucleic acid template, also Can comprise one section non-conterminous sequence of the primer sequence with starting template of 5 ' end. In other words, adjacent segment Be primer 3 ' one section sequence of end, the particular pilot sequence complementation of it and initial nucleic acid template, and non-conterminous Section is primer 5 ' one section sequence of end, it not with the guiding sequence complementation of initial nucleic acid template.

Herein, the initial nucleic acid template refers to contain or suspects in the sample that contains target sequence purifying or purifying not Nucleic acid. Therefore, the method can be used DNA, RNA or DNA:RNA heterozygote. Initial nucleic acid Template is not limited to finally be translated as DNA or the RNA of protein, also can comprise non-coding nuclear Non-coding nucleic acid sequence between acid or the coded sequence.

In order effectively to reduce the generation of non-specific guiding, the invention provides the sealing oligonucleotides, it Hybridize with primer with non-specific guiding sequence competition. Sealing oligonucleotides of the present invention " can not be done Primer for polymerase ", namely can not be as the synthetic starting point of extension products. The sealing oligonucleotides can not be done For the primer that extends reaction can be reached in the following way: remove or modify 3 ' terminal hydroxyl, as add the end End 3 '-two deoxidation nucleotides or 3 '-phosphorylation, 3 '-the amination modification, and near 3 ' end, connect Connect the big molecule such as rhodamine, reaction is extended in the catalysis of three-dimensional prevention polymerase. Extension products synthesizes Representative of conditions is referring to embodiment.

In most cases, when primer and its sealing oligonucleotide hybridization formation " primer: sealing oligonucleotide doublet ", 3 of primer '-end stretches out or to surpass its sealing oligonucleotide comparatively desirable, and can get rid of primer like this and carry out unnecessary extension along 5 ' → 3 ' direction.About this point, should be pointed out that primer: the formation of sealing oligonucleotide doublet is a reversible process, thereby under appropriate condition (as heating succeeded by cooling), doublet can repeat to unwind and reconnect.

" sealing oligonucleotide " speech general reference can with primer hybridization to prevent the oligonucleotide of non-specific guiding.Because sealing oligonucleotide primer corresponding with it is fully complementary, under suitable concn, it can stablize the primer that has neither part nor lot in the specificity guiding, thereby prevents non-specific guiding." fully complementary " refers to that the complementary degree of Nucleotide of two sequences should be enough to make hybridization to take place.Each primer all has the sealing oligonucleotide when being preferably in amplified reaction and beginning.

Persons of ordinary skill in the art may appreciate that complementary degree and concentration were interactional variable when hybridization took place two chains.Thereby the sealing oligonucleotide can primer height corresponding with it complementation, or the complementation of not corresponding with it primer height, as long as the concentration of oligonucleotide is enough to stop combining of primer and non-specific sequence.Usually nucleic acid samples is double-stranded, thereby must be separately double-stranded before extension products is synthetic.In following embodiment, thermally denature is used to the separately two strands of nucleic acid samples.Yet separately two strands also can be used other reagent as known in the art, comprises the enzyme reagent such as the RecBCD helicase, referring to (1981, Enzgme 14:233) such as Muskavitch.Primer and its sealing oligonucleotide can doublet or two monomeric forms adding nucleic acid samples.If primer and its sealing oligonucleotide add with the doublet form, make the step of nucleic acid samples sex change can make primer equally: sealing oligonucleotide doublet separately forms corresponding strand.

Chain separately after, reaction mixture places under the suitable hybridization conditions, promotes the primer and the homing sequence hybridization of its template to form primer: stamp complex, or with its sealing oligonucleotide hybridization, form primer: seal the oligonucleotide doublet.Suitable hybridization conditions is widely known by the people in the art, and some of them can be with reference to following examples.

In case after the hybridization of the homing sequence of primer and template, reaction mixture is placed under the suitable extension condition, makes primer as extension, i.e. the starting point of polyreaction.Primer not necessarily will carry out in two different reactions with the hybridization and the primer extension of template, thereby, comprehensively to hybridize required condition and extend required condition, hybridization can be carried out simultaneously with extension.Suitable extension condition is widely known by the people in the art, it is some controlled conditions (as temperature, pH and ionic strengths), in the presence of catalyzer such as archaeal dna polymerase, add Nucleotide, can induce extension products synthetic with the primer 3 ' end of nucleic acid-templated hybridization.Representational extension condition also can be referring to following examples.

The catalyzer of extension is archaeal dna polymerase normally, as the heat-resisting polymerase (Kong etc., J.Biol.Chem (1993) 268:1965) of Thermus aquaticus or Thermusthermophilus.Be to realize the present invention, very crucial a bit is, and polysaccharase can remove on the template and the oligonucleotide of the downstream hybridization of the sequence of primer hybridization.Under suitable extension condition, polysaccharase adds Nucleotide one by one to extending primer 3 ' end, remove may exist, with primer downstream sequence bonded sealing oligonucleotide, thereby synthetic a kind of extension products, the target complement sequence of its sequence and template, and comprise the sealing oligonucleotide sequence.

If template is a double-strandednucleic acid, then at first synthetic two kinds of extension products, each extension products is corresponding to a chain of target sequence.New synthetic extension products is not only the replica of target sequence, and comprises homing sequence, and this homing sequence may contain or not have one section non-conterminous sequence.New synthetic extension products separates with the starting template chain subsequently, and as the template of synthesizing other extension products.After this, primer is if contain and the non-conterminous fragment of the homing sequence of starting template, and then this fragment also is replicated, and is incorporated among the extension products.Should point out that each extension products can be used as template again in follow-up extension, it is made up of two covalently bound parts: (i) whole primer, comprise the non-conterminous fragment that may exist, and (ii) be complementary to the amplifying nucleic acid sequence of a chain of target sequence.Double-stranded separately, hybridization and extension/displacement finish a circulation having formed amplified reaction.Thereby the concentration of extension products increases with index speed with the increase of amplification cycles.

In a preferred embodiment, primer and its sealing oligonucleotide be mark respectively, and as rolling into a ball mark with donor fluorophore and acceptor fluorescence respectively, they are extremely approaching mutually by hybridization, thereby the resonance energy transfer can take place.Along with the carrying out of extension, because labeled primer constantly is impregnated in the extension products, the labeled primer that can access constantly reduces, and this has just reduced the quantity that can seal the labeled primer of oligonucleotide hybridization with mark.Along with repeating of extension.Produced can with the unmarked extension products of primer bonded, it and mark sealing competitive oligonucleotide and primer hybridization.Thereby in this preferred embodiment, the sealing oligonucleotide serves a dual purpose: (1) reduces or eliminates non-specific guiding as the sealing molecule, and (2) participate in testing process as signal probe.

When fluorophore is used to realize when of the present invention, donor and acceptor fluorescence group are connected on the corresponding primer with it of sealing oligonucleotide, like this, when sealing oligonucleotide and primer when mutual cross forms doublet mutually, the distance between them is within the resonance energy metastasis range.Preferably separate between the fluorophore by at least one Nucleotide.But distance is not greater than 100A (Clegg, Methods ib Enzymology 211:253).Fluorophore is connected on the inner core thuja acid also more satisfactory.If be necessary, can use two above fluorophores.Should also be noted that fluorophore can be identical or different.The excitation spectrum of the emmission spectrum of donor fluorophore and acceptor fluorescence group overlapping makes the energy transfer can take place between them.Thereby when primer: sealing oligonucleotide doublet sex change or separately the time (as in the extension) has just produced the signal variation because energy shifts interruption.

If need, can at first prepare a kind of sealing oligonucleotide of fluorophore mark, it and all fully complementations (complementarity that promptly has 5 base pairings at least) of target sequence of primer or starting template.General detection doublet is made up of the oligonucleotide of fluorophore mark sealing oligonucleotide and another kind, wherein seals the oligonucleotide and the second oligonucleotide complementation, and stretches out 3 ' end of second oligonucleotide.Mixing specific primer and starting template with before carrying out extension, the sealing oligonucleotide becomes the complementary strand of primer by ligation.In ligation, 3 of second oligonucleotide ' end is covalently bound with 5 of primer ' end.In other words, " general detection doublet " is meant by sealing oligonucleotide and not right as the oligonucleotide that the oligonucleotide of primer forms, apparent with its complementary, and two kinds of (or more kinds of) fluorophores of general detection doublet are must extreme approaching.In a particularly preferred embodiment, donor fluorophore and acceptor fluorescence group are connected on the corresponding oligonucleotide of general detection doublet of weak stability, and primer is connected in the comparatively stable doublet-primer of formation on the doublet subsequently.Thereby the energy transfer efficiency between two kinds of fluorophores has improved.

Different with general detection doublet, special detection doublet need not to modify and promptly can be used for amplification/detection target nucleic acid, and referring to Fig. 1, filled circles and closed square are represented two kinds of fluorophores, the invalid 3 ' end of open circles representative sealing oligonucleotide.It should be noted that in the embodiment depicted in fig. 1 labeled primer comprises not adjacent with its homing sequence fragment.And preferential amplification of the asymmetric amplification of carrying out in advance and labeled primer bonded chain are so that can improve susceptibility.Asymmetric amplification can cause by the following method: the inequality primer is provided, and the excessive and another kind of primer of promptly a kind of primer is limited the quantity of, or provides and the different primer of the complementary degree of particular pilot sequence.Other factors, the difference as the synthesis rate of each template strand may also help asymmetric amplification.The best primer ratio of asymmetric amplification can determine by rule of thumb (Shyamala etc., 1989.J.Baceriol.171:1602).

Another kind of " special detection doublet " that method of the present invention is used sees Fig. 2.In this embodiment, used two kinds of unlabelled special detection doublets (promptly two the sealing oligonucleotide: primer to).Two primers all do not have and the non-conterminous fragment of its primer sequence.Equally, the invalid 3 ' end of open circles representative sealing oligonucleotide.It should be noted that this there is shown the significant process of removing downstream sealing oligonucleotide, and not shown among Fig. 1.

Method of the present invention can be carried out on a kind of instrument, and on this instrument, the amplification of nucleic acid-templated middle target sequence can be carried out simultaneously with the target sequence of measuring amplification, need not separating step.This instrument comprises: (1) holds the oligonucleotide container of oligonucleotide; (2) hold the reaction vessel of nucleic acid samples, the mode of connection of this reaction vessel and oligonucleotide container makes the inclusion in the oligonucleotide container can enter reaction vessel automatically; (3) controller of mixing oligonucleotide and sample; (4) temperature regulator of control reaction vessel temperature; (5) irradiation source; (6) measure the radiating detector that sends by reaction vessel.The preferred reaction container is one of porous microtiter plate hole.Usually, irradiation source is for producing the device of the photon of selecting wavelength, and this device is that those of ordinary skill in the art knows, and commercially available (as tungsten lamp or ion laser lamp).Detector is for being converted to photon energy the device of electrical signal, and same, this device also is widely known by the people, and can be provided by numerous suppliers, and the example of this device comprises photomultiplier and photonic semiconductor detector.

Preferred especially this instrument also comprises a data treater, to handle the signal data that detector receives.This instrument also can comprise a kind of controlling organization, and when detector detects predetermined radiation level, when promptly being equivalent to previously selected level of amplification, this controlling organization stops amplified reaction automatically.Thereby this instrument provides the method for a kind of " online " monitoring amplified reaction, and this method is similar to the method for the online monitoring tunning that extensive recombinant protein uses in synthetic.Thereby homogeneous phase amplification and detection method and disclosed herein instrument are united use, can eliminate the pollution of reaction vessel, because there is no need by sampling in the reaction mixture to determine the degree of target sequence amplification.

Can believe, need not to further specify that those skilled in the art can make full use of the present invention according to description herein.Thereby following specific embodiments is interpreted as just illustration, rather than by any way all the other open parts is limited.

Example I: the effect of sealing oligonucleotide aspect the non-specific guiding of minimizing

For the influence of sealing oligonucleotide to boot efficiency is described, the 127bp fragment of L sry gene (Sinclair etc., 1990, Nature, 346: 240) increases under existence of sealing oligonucleotide or non-existent situation.The target-seeking primer sequence is: primer (A) 5 '-AACTC AGAGA TCAGC AAGCAGCTGG GATAC-3 ', primer (B) 5 '-ACTTA TAATT _TRCGGGT ATTTC T _TRCTCTGTGCA-3 '.T _TRRepresent an amino of puting together by texas Red-C6-dT residue (GlennResearch, Sterling.VA), and texas Red be according to the method for supplier suggestion put together in primer (B) go up (Molecular Probes, inc.Eugene.ORU.S.A).24 base complementrities of used sealing oligonucleotide and primer (A), its sequence are 5 '-CAGCT GCTTG CTGAT CTCTG AGTTL-3 ', wherein, " L " expression 3 '-the amine end (3 '-amine-ON CPG, Clontech, Palo Alto, CA).

All oligonucleotide are by Operon technologies, and (Alameda CA) synthesizes, then electrophoresis purifying on 8M urea/15% acrylamide gel Inc..The special band of oligonucleotide is got off by electrophoresis elution, and is undertaken quantitatively by the uv-absorbing of surveying the 260nm place.

Male body genomic dna as target DNA is to adopt the DTAB/CTAB method by the (Gustinicich etc. that extract in the whole blood, 1991.Biotechniques 11:298), the photometry of 260nm place absorbs to be carried out quantitatively, with TE damping fluid (10mM Tris-HCl, 1mM EDTA, pH8.0) connect dilution, adopt 50ng/ μ l yeast tRNA as carrier.

AG-9600 Thermal Station is adopted in amplification, and (MJ Research, Watertown MA), carry out on 96 hole polycarbonate microtiter plates.The reaction system volume is 10 μ l, contains 50mM Tris-HCl, pH8.7,40mM KCl, 5mM NH ₄Cl, 2mM DTT, 0.1%Triton X-100,100 μ M dNTP, (Carballeira etc. are 1990.Biotechniques9:276-281) with the target DNA (10 of different amounts for 0.3 Tth of unit archaeal dna polymerase ², 10 ³With 10 ⁴), adding each 30ng of every kind of primer, sealing oligonucleotide and primer equivalent add or do not add.With yeast tRNA and salmon sperm DNA as negative control.Carry out altogether 35 thermal cyclings (94 ℃ 25 seconds, 60 ℃ 20 seconds, 72 ℃ 40 seconds).Subsequently, adopt 9% acrylamide gel electrophoresis to carry out sample analysis, electrophoretic buffer adopts tbe buffer liquid, and voltage is 10v/cm, and the molecular weight marker of gel electrophoresis is the pBR322 DNA of HpaII digestion.Gel is taken a picture under UV-light with the ethidium bromide rapid dyeing, and photo as shown in Figure 3.

Referring to Fig. 3, the implication of each swimming lane all is illustrated on the gel.Wherein, tRNA represents yeast tRNA, and SS represents salmon sperm DNA, and M represents molecular weight marker, the amount of digitized representation target molecule, and symbol "+" or "-" expression are with or without the sealing oligonucleotide.Have in the reaction system of sealing oligonucleotide, the brightness of 127 bp SRY distinguished sequence bands is higher, and is particularly evident when target DNA dosage is low.Otherwise, not adding in the reaction system of sealing oligonucleotide, the brightness of primer binary band is higher.Thereby the amplification efficiency of specific target sequence directly is subjected to non-specific guiding, and as the influence of the annoyance level that forms the primer binary, this primer binary is obviously more in not containing the reaction system of sealing oligonucleotide.

Example II: adopt the amplification of the sealing oligonucleotide monitoring target nucleic acid of fluorophore mark

Present embodiment explanation sealing oligonucleotide not only can be used to reduce non-specific guiding, and with behind the fluorophore mark, also can be used to monitor amplification procedure.Used target DNA in the present embodiment, sealing oligonucleotide and primer basically with example I in identical, has only indication probe difference, 20 base complementrities of this probe and primer (B), its sequence is 5 '-AGAGA GAAAT ACCCG AATTAL-3 ', its participates in the fluorescence energy transfer with primer (B)." L " expression 3 '-the amine end (3 '-amine-ONCPG, Clontech, Palo Alto CA).Behind the purifying, the indication probe is puted together with fluorescein again, puts together that (OR.USA) Jian Yi method is carried out for Molecular Probes, Inc.Eugene by supplier.

Amplification is to adopt a MW-1 thermal cycler (Techne Inc.Cambridge, England), (Techne Inc, Cambridge carry out on England) at 96 hole polycarbonate microtiter plates.The reaction system volume is 20 μ l, contains 50mM Tris-HCl, pH8.7,40mM KCl, 5mM NH ₄Cl, 2mMDTT, 0.1%Triton X-100,100 μ M dNTP, 0.6 Tth of unit archaeal dna polymerase, 10 ²Individual target DNA copy adds each 20ng of every kind of primer, and sealing oligonucleotide and primer equivalent add or do not add.Salmon sperm DNA is as negative control.Carry out altogether 24 thermal cyclings (96 ℃ 42 seconds, 53 ℃ 42 seconds, 72 ℃ 60 seconds).In each reaction system, add the indication probe subsequently.The 25th circulation time, (Model 7600, Cambridge Technologies to use the micro plate photofluorometer, Inc., Watertown, MA, USA) each reaction system is carried out fluorescence reading first at the 630nm place, subsequently at the 30th circulation and the 35th reading once more that circulates.The fluorescence reading is plotted curve, and shown in Figure 4.

The bar graph explanation adds or does not add two different round-robin fluorescent signal intensity of variations that seal oligonucleotide among Fig. 4 when the amplification beginning.Fluorescent signal descends and shows primer: sealing oligonucleotide doublet unwinds, and when not having non-specific guiding, fluorescent signal decline is directly proportional with the degree that primer mixes the target sequence of amplification.Contrast is designated as " amplification " as fluorescent signal not add the reaction that T goes out archaeal dna polymerase.To containing 10 ²The parallel test of sample of copy SRY sequence and salmon sperm DNA is to observe special signal of target DNA and non-special signal.As shown in FIG., in the amplification later stage (the 35th circulation), the existence of sealing oligonucleotide reduces non-specific guiding, and this can be led by the difference of change in fluorescence between two salmon sperm DNA contrasts and find out, contains or do not contain the sealing oligonucleotide respectively in above-mentioned two contrasts.

EXAMPLE III: general detection doublet is connected the raising energy to be shifted with primer

In the present embodiment, two cover general oligonucleotides are used to illustrate that the energy that ligation causes shifts raising.In the ligation, as template, its complementary oligonucleotide is connected with primer with the sealing oligonucleotide.

Synthetic, the purifying of all oligonucleotide and to be connected with fluorophore all be to carry out according to the described method of previous embodiment.In the ligation, its complementary oligonucleotide and the primer that 200ng are sealed oligonucleotide and equimolar amount add in the 20 μ l reaction mixtures, contain 0.15 T of unit in this reaction mixture ₄Ligase enzyme (NewEngland Biolab, Beverly, MA), 0.5 T of unit ₄Polynueleotide kinase (New EnglandBiolab, Beverly, MA), 1mM ATP and damping fluid (70mM Tris HCl pH=7.6,10mMMgCl ₂And 5mMDTT).Ligation carries out twice, is reflected to carry out 1 hour 30 minutes under 40 ℃, by heating 10 minutes termination reactions at 95 ℃.Before and after each reaction, respectively get a equivalent reaction mixture, survey the emitting fluorescence intensity at its 630nm place under the 495nm optical excitation.

First cover: the sealing oligonucleotide sequence is 5 '-T _TRTTTT GAACA GGCCT TGT _TRG-3 ', its complementary oligonucleotide sequence is 5 '-CAAGG CCT _FG-3 ', primer sequence be 5 '-TTCAA A CCTG TGCTT AGGGC ACTG-3 ', base that bottom line marks and homing sequence complementation.T _TRAmino-C6-dT residue that the expression texas Red is puted together, T _FAmino-C6-dT residue that the expression fluorescein is puted together.Example I and II are seen in the method for attachment of they and oligonucleotide.

The fluorescence reading of reaction mixture is 900 ± 23 before the ligation, and the reading after the ligation is 1336 ± 211, thereby the energy transfer has improved 1.48 times after connecting in the first cover doublet.

Second cover: the sealing oligonucleotide sequence is 5 '-T _TRTGGT CTCGA CCTCC TTGT _TRG-3 ', its complementary oligonucleotide sequence is 5 '-CAAGG AGGT _FC G-3 ', primer sequence be 5 '-AGACC A CCTG TGCTT AGGGC ACTG-3 ', base that bottom line marks and homing sequence complementation.

Before the ligation, the fluorescence reading of reaction mixture is 1489 ± 58, and reading is 3019 ± 94 after the ligation, thereby, connect back energy transfer in the second cover doublet and improved 2.03 times.

EXAMPLE IV: use the asymmetric amplification that detects doublet monitoring target nucleic acid

Be the application of explanation detection doublet, we use: (A) primer: sealing oligonucleotide doublet, or the general detection doublet monitoring amplification procedure that (B) is connected with primer.In the doublet (A), sealing oligonucleotide and primer and initial target sequence are all complementary.In the doublet (B), primer is connected on the complementary oligonucleotide of sealing oligonucleotide, sets up complementary relationship between primer and sealing oligonucleotide.Doublet (A) and (B) all be used for same amplified reaction.With the acute medullary cell leukemia of people breaking point correlated series (AML-1) and people's x-chromosome specificity glaze former (ameloginen) fragment (AMG-X) as the amplified target sequence, so that the application of above-mentioned doublet to be described. Primer: the preparation of sealing oligonucleotide doublet

Doublet (A): the sequence that is used for the primer of AML-1 and sealing oligonucleotide is respectively 5 '-ACGGG GA TAC GCATC ACAAC AA-3 ' and 5 '- TTGAT GCGTA TCCCCGT TT-3 '; The sequence that is used for the primer of AMG-X and sealing oligonucleotide is respectively 5 '-AGACTGAG TC AGAGT GGCCA GGC-3 ' and 5 '- TCCAC TCTGA CTCAG TCT TA-3 '.Put together with fluorescein and texas Red respectively the position that marks with bottom line on primer and the sealing oligonucleotide.3 ' end of sealing oligonucleotide is a dideoxy nucleotide, thereby can't extend.

Doublet (B): the primer sequence that is used for AML-1 is 5 '- GGGAC GCACG GGGAAACGCA TCACA ACAA-3 '; The primer sequence that is used for AMG-X is 5 '- GGGAC GCAGACTGAG TCAGAGTGGC CAGGC-3 '.Every kind of primer all is connected on the detection doublet that is similar in the EXAMPLE III.The AML-1 primer is connected back energy transfer with the AMG-X primer and has improved 2.62 and 2.42 times respectively with doublet. Asymmetric amplification and detection

The excessive primer of AML-1 target sequence is 5 '-TGTTT GCAGG GTCCT AACTCAATCG-3 ', the primer of limiting the quantity of is 5 '-GCGGC GTGAA GCGGC GGCTC G-3 '; The excessive primer of AMG-X target sequence is 5 '-TGATG GTTGG CCTCAAGCCT GTG-3 ', the primer of limiting the quantity of is 5 '-TGGGATAGAACCAAG CTGGT CAG-3 '.

As target sequence, this DNA adopts the method for example I by extracting in the whole blood with human male's genomic dna.

Before adding the primer doublet, with 20 circulations of the asymmetric amplification of target sequence.During asymmetric amplification, excessive primer is 7.5 (1.2 μ m are than 0.16 μ m) with the primer ratio of limiting the quantity of, and the reaction system volume is 10 μ l, contains 1 T of unit _ThArchaeal dna polymerase, 50mM Tris HCl, 40mM KCl, 5mM NH ₄Cl, 100 μ m dNTP, 5mM MgCl ₂And 0.1%TritonX-100.Cycling condition is identical with example I.AG-9600 Silver Block Thermal Station is adopted in reaction, and (MA), (Costar, Cambridge carry out on MA) at 96 hole polycarbonate micro plates for MJ Research, Watertown.

At room temperature every kind of primer doublet 4ng is added in the reacting hole to begin amplification and signal detection process.The 21st circulation time measured 630nm place fluorescent emission, first as the baseline of typical curve under 485nm excites.Measure once more the 28th circulation subsequently, measure every a circulation later on.

The 30th round-robin detected result is shown among Fig. 5, and The above results has illustrated doublet (A) and (B) quantitative resolving power and detection sensitivity.As shown in FIG., fluorescence intensity decline has reflected the amount of initial target sequence.

Other embodiment

According to above description, those skilled in the art is easy to determine inner characteristic of the present invention, and, under the prerequisite that does not exceed design of the present invention and scope, can make the present invention and changing and adjustment, so that it adapts to various application and condition, thereby, also comprise other embodiment in the claim.

Claims (30)

1, a kind of method that detects target nucleic acid, this method may further comprise the steps:

Use polysaccharase, first primer and second primer in the presence of a kind of oligonucleotide amplifying target nucleic acid with the acquisition amplified production, wherein first primer has or does not have and the non-conterminous fragment of first homing sequence, second primer has or does not have and the non-conterminous fragment of second homing sequence, described oligonucleotide can not be as the primer of described polysaccharase, but this oligonucleotide has at least 5 successive Nucleotide to be complementary at least 5 successive Nucleotide of described first primer fully; With

Detect the existence of target nucleic acid by monitoring above-mentioned amplification.

2, method as claimed in claim 1 is characterized in that described detection step is at the amplified production of monitoring target nucleic acid behind the electrophoresis on gel.

3, method as claimed in claim 1, it is characterized in that first fluorophore and described first primer covalently bound and second fluorophore and described oligonucleotide is covalently bound, and it is a kind of in described first and second fluorophores as the donor fluorophore, another kind of as acceptor fluorescence group, make when described first primer and described oligonucleotide hybridization, described donor fluorophore and described acceptor fluorescence group are extremely approaching, resonance energy can take place each other shift; And, described detection step is when with exciting light described donor fluorophore being carried out irradiation, the fluorescent emission of described acceptor fluorescence group changed monitor, described variation has reflected that first primer and described oligonucleotide unwind and mix degree in the amplified production of target nucleic acid.

4, method as claimed in claim 1 is characterized in that described first primer has and the non-conterminous fragment of described first homing sequence.

5, method as claimed in claim 4 is characterized in that described oligonucleotide has at least 5 successive Nucleotide in the described non-conterminous fragment that at least 5 successive Nucleotide are complementary to described first primer fully.

6, method as claimed in claim 1 is characterized in that described first primer does not have and the non-conterminous fragment of described first homing sequence.

7, method as claimed in claim 1, it is characterized in that described amplification step is to carry out in the presence of another kind of oligonucleotide, this oligonucleotide can not be as the primer of described polysaccharase, wherein, described another kind of oligonucleotide has at least 5 successive Nucleotide that at least 5 successive Nucleotide are complementary to described second primer fully.

8, method as claimed in claim 1 is characterized in that described first primer, second primer and oligonucleotide respectively contain 10-50 Nucleotide.

9, method as claimed in claim 8 is characterized in that described first primer, second primer and oligonucleotide respectively contain 15-40 Nucleotide.

10, method as claimed in claim 1 is characterized in that described oligonucleotide has at least 8 successive Nucleotide that at least 8 successive Nucleotide are complementary to described first primer fully.

11, method as claimed in claim 1, the concentration that it is characterized in that described oligonucleotide are 0.3～5.0 times of described first primer.

12, as the method for claim 11, the concentration that it is characterized in that described oligonucleotide is 0.5～2.5 times of described first primer.

13,, it is characterized in that described first primer, second primer and oligonucleotide respectively contain 10-50 Nucleotide as the method for claim 12.

14,, it is characterized in that described oligonucleotide has at least 8 successive Nucleotide that at least 8 successive Nucleotide are complementary to described first primer fully as the method for claim 12.

15,, it is characterized in that described oligonucleotide has at least 8 successive Nucleotide that at least 8 successive Nucleotide are complementary to described first primer fully as the method for claim 13.

16, as the method for claim 15, it is characterized in that first fluorophore and described first primer covalently bound and second fluorophore and described oligonucleotide is covalently bound, and it is a kind of in described first and second fluorophores as the donor fluorophore, another kind of as acceptor fluorescence group, make when described first primer and oligonucleotide hybridization, described donor fluorophore and acceptor fluorescence group are extremely approaching, resonance energy can take place each other shift; And described detection step is when with exciting light the donor fluorophore being carried out irradiation, the fluorescent emission of acceptor fluorescence group is changed monitor, and this variation has reflected that first primer and described oligonucleotide unwind and mix degree in the amplified production of target nucleic acid.

17, a kind of test kit that is used to detect target nucleic acid comprises:

First primer, it has or does not have and the non-conterminous fragment of first homing sequence; Second primer, it has or does not have and the non-conterminous fragment of second homing sequence; Described first primer and second primer are used to amplifying target nucleic acid with a kind of polysaccharase; With

A kind of oligonucleotide, it can not be as the primer of described polysaccharase, but has at least 5 successive Nucleotide that at least 5 successive Nucleotide are complementary to described first primer fully;

Wherein, described first primer, second primer and oligonucleotide respectively contain 10-50 Nucleotide.

18,, it is characterized in that described first primer and oligonucleotide provide with the doublet form as the test kit of claim 17.

19,, it is characterized in that described oligonucleotide has at least 8 successive Nucleotide that at least 8 successive Nucleotide are complementary to described first primer fully as the test kit of claim 17.

20,, it is characterized in that described first primer, second primer and oligonucleotide respectively contain 15-40 Nucleotide as the test kit of claim 19.

21, as the test kit of claim 19, also comprise a kind of polysaccharase.

22, as the test kit of claim 19, also comprise another kind of oligonucleotide, it can not be as the primer of described polysaccharase, but it contains 10-50 Nucleotide, and has at least 8 successive Nucleotide that at least 8 successive Nucleotide are complementary to described second primer fully.

23, as the test kit of claim 19, it is characterized in that two kinds of fluorophores are covalently attached to respectively on described first primer and the oligonucleotide, a kind of in two kinds of fluorophores as the donor fluorophore, another kind of as acceptor fluorescence group, like this, when described first primer and oligonucleotide hybridization, described donor fluorophore and acceptor fluorescence group are extremely approaching, resonance energy can take place each other shift.

24, a kind of test kit that is used to detect target nucleic acid comprises:

Contain first oligonucleotide of 10-50 Nucleotide, first fluorophore is thereon covalently bound, and this first oligonucleotide can not be as the primer of polysaccharase when amplifying target nucleic acid; With

Contain second oligonucleotide of 5-30 Nucleotide, second fluorophore is thereon covalently bound, and this second oligonucleotide has free 3 ' OH, and can pass through at least 5 complete complementary nucleotide pairs of successive and described first oligonucleotide hybridization; Wherein, described first oligonucleotide has the overhang that exceeds 1-12 Nucleotide than described second oligonucleotide, 3 ' end, and, described first and second fluorophores are extremely approaching when described first oligonucleotide and described second oligonucleotide hybridization, thereby resonance energy can take place between these two kinds of fluorophores to be shifted, a kind of in wherein said two kinds of fluorophores, another kind of as acceptor fluorescence group as the donor fluorophore.

25, as the test kit of claim 24, it is characterized in that described first oligonucleotide contains 15-40 Nucleotide, and have the overhang that exceeds 4-8 Nucleotide than described second oligonucleotide, 3 ' end, this overhang can pass through at least 8 complete complementary nucleotide pairs of successive and described first oligonucleotide hybridization.

26,, it is characterized in that described first and second oligonucleotide provide with the doublet form as the test kit of claim 25.

27, as the test kit of claim 25, also comprise a kind of ligase enzyme.

28,, also comprise a kind of kinases or a kind of polysaccharase with 5 ' → 3 ' exonuclease activity as the test kit of claim 25.

29,, also comprise a kind of kinases or a kind of polysaccharase with 5 ' → 3 ' exonuclease activity as the test kit of claim 27.

30,, also comprise a kind of polysaccharase that has or do not have 5 ' → 3 ' exonuclease activity as the test kit of claim 25.

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