CN100393875C - Method for synthesizing nucleic acid - Google Patents

Abstract

An oligonucleotide having a novel structure and a method for synthesizing a nucleic acid by using the same as a primer. In the 5'-side of the primer, this oligonucleotide is provided with a base sequence which is substantially identical with the region to be synthesized by using this primer as a starting point of the synthesis. Thus, a nucleic acid can be synthesized on the basis of an isothermal reaction with the use of a simple reagent constitution. Also, provision is made of a method for synthesizing a nucleic acid having a high specificity based on the above method for synthesizing a nucleic acid.

Description

The method of nucleic acid

Invention field

The present invention relates to the synthetic method of the nucleic acid that constitutes by specific nucleotide sequence, a kind of method of useful amplification of nucleic acid.

Background of invention

But analytical procedure direct analysis hereditary feature based on the nucleotide sequence complementarity.Therefore, this analysis is a kind of evaluation genetic diseases, canceration, very strong method such as microorganism.And, detection to as if gene self, so under some situation consuming time and loaded down with trivial details operating process as just can omit in cultivating.

Yet,, therefore must increase to target gene self or its detection signal when the sample gene dosage that hits generally is difficult for when considerably less detecting.As the method for amplified target gene, PCR (polymerase chain reaction) method known for everyone (Science, 230, 1350-1354,1985).At present, PCR method is the most general technological method of amplification in vitro nucleotide sequence.The exponential amplification result makes it have highly sensitive advantage, so this method has been established a kind of extraordinary detection approach firmly.In addition, because the amplified production that is recovered to can be DNA, therefore as a kind of for example important tool of gene clone and structures shape of gene engineering of supporting, this method has obtained widespread use.Yet PCR method obviously has following problem: must be with special temperature regulator in the actually operating; The exponential form of amplified reaction rises and causes having problems in quantitatively; Sample and reaction soln are vulnerable to external contamination, and the nucleic acid of sneaking into is moved as Template Error ground.

Genomic information increasing, single nucleotide polymorphism (SNPs) analysis progressively has been subjected to attention.Make its nucleotide sequence comprise SNPs by the design primer, it is feasible relying on PCR to detect SNPs.Just, whether exist with primer complementary nucleotide sequence can by whether existing the decision of reaction product to draw deduction.Yet in case the synthetic complementary strand of accidental mistake among the PCR, this product moves with template in subsequent reaction, the result that will make the mistake.In the practical application, allegedly only a base is different from the very difficult strict PCR that control for the primer end.Therefore, must improve specificity is applied in the detection of SNPs PCR.

On the one hand, in the reality also in the method for using the ligase enzyme nucleic acid.LCR method (ligase chain reaction (LCR) (ligase chain reaction), Laffler TG; Garrino JJ; Marshall RL; Ann.Biol.Clin. (Paris), 51: 9,821-6,1993) be based on such reaction, two adjacent probes and target sequence hybridization and to pass through ligase enzyme interconnection in this reaction.Described two probes can not be connected under the situation of target nucleotide sequences lacking, and the existence that therefore connects product is the symbol that target nucleotide sequences exists.The LCR method also needs controlled temperature to be used for separating complementary strand from template, runs into problem identical in the PCR method.For LCR, have by being increased in the step that breach is provided between adjacent probe and utilizes the archaeal dna polymerase fill up the gap to improve the report of specificity method.This improves one's methods desired is specific improvement, yet, so owing to need controlled temperature still to have problems.In addition, use extra enzyme to cause expense to increase.

Be called SDA method (strand displacement amplification, strand displacement amplification) [Proc.Natl.Acad.Sci.USA, 89, 392-396,1992] [Nucleic.Acid Res., 20, 1691-1696,1992] also be the method that known amplification has the template DNA of sequence and target complement sequence.In the SDA method, when replacing the sequence of double-stranded 5 '-side, with specific archaeal dna polymerase from beginning to synthesize complementary strand with certain nucleotide sequence 3 '-side complementary primer.Simple expression 5 '-side and 3 '-side refers to template strand among the present invention, because new synthetic complementary strand has been replaced the two strands of 5 '-side, claims that this technology is the SDA method.The restriction enzyme recognition sequence is inserted in advance in the anneal sequence as primer and just can removes temperature change steps necessary in the PCR method in the SDA method.Promptly the otch that generates by restriction enzyme is supplied with the synthetic starting point of 3 '-OH base as complementary strand, and first synthetic complementary strand then is used for following complementary strand as template once more and synthesizes by the synthetic strand that is released of strand displacement.Like this, in the SDA method, just do not need necessary complicated temperature control in the PCR method.

Yet in the SDA method,, also to use the restriction enzyme that generates otch except strand displacement type archaeal dna polymerase.Needing to use extra enzyme is the major cause that causes than high cost.In addition, because restriction enzyme is not to be used for disconnecting two chains, but for producing otch (just only disconnecting wherein chain), dNTP derivative for example α-sulfo-dNTP is used as the synthetic substrate and makes other chain can resist the digestion of this enzyme.Therefore, the SDA amplified production is different with the natural acid structure, and to rupturing with restriction enzyme or amplified production being applied to existence restriction in the gene clone.This respect also is to cause the expense major reason for higher.In addition, when the SDA method was applied in unknown nucleotide sequence, the nucleotide sequence identical with the restriction enzyme recognition sequence that is used for introducing breach may be present in the district that will be synthesized.In this case, might stop the synthetic of complete complementary strand.

NASBA (amplification (nucleic acid sequence-based amplification) based on nucleotide sequence also claims TMA/ transcriptive intermediate amplification method) also is the amplification of nucleic acid method known to us, does not wherein need complicated temperature control.NASBA is a reactive system, wherein DNA is being template by archaeal dna polymerase with target RNA, adding has the probe of T7 promotor and is synthesized, second probe enters two strands is generated product, be that template is passed through the RNA (Nature that the T7 rna polymerase transcribe increases a large amount of then with the double-stranded DNA that generates 350, 91-92,1991).NASBA needs some thermally denature step to form up to double-stranded DNA, but subsequent responsive transcription is undertaken by the T7 RNA polymerase under isothermal condition.Yet must make up for example ThermoScript II with plurality of enzymes, RNase H, archaeal dna polymerase and T7 RNA polymerase, then the combination of plurality of enzymes similar to SDA this be disadvantageous for expense.And owing to set the reaction conditions complexity of plurality of enzymes, this method is difficult to be promoted as general analytical procedure.In the known nucleic acid amplification reaction, also exist complicated as mentioned above temperature to control and need use the problem of plurality of enzymes.

To the reaction of these known nucleic acids, seldom relevant for further improving nucleic acid synthetic efficient and don't losing the report of the trial of specificity or expense.For example be called RCA (rolling circle amplification, rolling-circle amplification) in the method, demonstration has a series of nucleotide sequences and padlock-probe (padlock probe) complementary single stranded DNA can be by synthetic (the Paul M.Lizardi et al. of successive in the presence of target nucleotide, Nature Genetics 19, 225-232, July, 1998).In RCA, use and have the special construction padlock-probe, wherein in LCR the oligonucleotide of a chain each 5 '-and 3 '-terminally constitute adjacent probe.Be that template is passed through the reaction in conjunction with the synthetic strand displacement type complementary strand of polysaccharase catalysis then with the padlock-probe, the successive reaction of synthetic complementary strand is started, and this template is connected in the presence of target nucleotide sequences and cyclisation.The single-chain nucleic acid of Sheng Chenging has a kind of multiple successive structure thus, and each district is made up of identical Nucleotide.Primer further is annealed and makes this single-chain nucleic acid synthesize its complementary strand, thereby realizes highly amplification.But still there is the problem that needs plurality of enzymes.And the complementary strand synthetic starts and to depend on the reaction that connects two proximities, and its specificity basically with LCR in identical.

For 3 '-OH is provided, in currently known methods, offers nucleotide sequence and its complementary sequence, and form hairpin loop (Gene 71,29-40,1988) endways at 3 '-end.With target sequence from beginning to form the single-chain nucleic acid that constitutes by complementary nucleotide sequence at the hairpin loop place as synthesizing of template complementary strand.For example, same chain end generation annealed structure realized with being connected of complementary nucleotide sequence in PCT/FR95/00891.Yet terminal elimination is with this complementary strand paired base and reconstruct base pairing on same chain again, and this step is necessary in this method.A kind of little equilibrium state that relates to the mutual complementary nucleotide sequence of base pair paired end is depended in the operation in this step according to estimates.Just, and complementary strand paired base and same chain on keep a kind of equilibrium state between the paired base.Utilize this equilibrium state and only with same chain in Nucleotide annealed chain be complementary strand synthetic starting point.So, think and should set strict reaction conditions to obtain high reaction efficiency.Further, in the tip technology, primer self forms a kind of structure of ring.Therefore, in case form the primer dipolymer, no matter whether have target nucleotide sequences, amplified reaction can begin automatically, so will synthesize non-specific product.This is serious problem.In addition, the consumption of primer causes the amplification efficiency of required reaction to reduce in the formation of primer dipolymer and the ensuing non-specific building-up reactions.

In addition, the district that has utilization not serve as the archaeal dna polymerase template realize can with the report (EP713922) of same chain annealed 3 '-end structure.Owing to generate the dipolymer primer, the possibility aspect of the utilization of dynamic equilibrium and non-specific building-up reactions endways, there be the problem identical with PCT/FR95/00891 recited above in this report.In addition, can not should be made into primer for archaeal dna polymerase provides the district of template.

In addition, above-mentioned NASBA principle is applied to often utilize the terminal promoter region (JP-A 5-211873) that the hairpin structure oligonucleotide provides two strands that has in the various amplification of signal reactions.Yet these are not that those are the synthetic technology that 3 '-OH is provided continuously of complementary strand.And, in JP-A 10-510161 (WO96/17079) with hairpin ring structure to reach the purpose of the dna profiling that obtains rna polymerase transcribe, this hairpin ring structure is annealed at same chain 3 '-end.Template is increased to the DNA reverse transcription by being transcribed into RNA and RNA.Yet in this method, a large amount of enzyme of reactive system debond just can not be configured.

Summary of the invention

The method that the purpose of this invention is to provide a kind of nucleic acid, this method is based on a kind of new principle.Purpose more specifically provides a kind of cost effective method that can realize relying on the efficient nucleic acid of sequence.Just, the purpose of this invention is to provide by under a kind of single enzyme even isothermal condition, finishing the method that nucleic acid is synthetic and increase.Another object of the present invention provides a kind of nucleic acid synthetic method, and this method can realize the high specific that is difficult to reach with known nucleic acid building-up reactions principle also providing a kind of usefulness this synthetic method amplification of nucleic acid method.

The inventor concentrates on this to attention in fact, utilizes the complementary strand of polysaccharase catalysis strand displacement type synthetic and do not need complicated temperature control, is of value to the synthetic of nucleic acid.This archaeal dna polymerase is the enzyme of using among SDA and the RCA.Yet, promptly use such enzyme, in based on the currently known methods of primer, always need another kind of enzyme reaction to provide 3 '-OH as synthetic starting point, for example SDA.

Under these situations, the inventor uses the supply that 3 '-OH has been discussed with the diverse method of currently known methods, found that the oligonucleotide that has ad hoc structure by utilization, does not need any extra enzyme reaction 3 '-OH just can be provided, and draws the present invention thus.Promptly the present invention relates to the method for nucleic acid, by the new oligonucleotide with the described method of methods and applications of described nucleic acid synthetic method amplification of nucleic acid, as described below:

(1) synthesize the method for the nucleic acid of the complementary nucleotide sequence that has alternately connection on one bar chain, described method comprises:

A) provide a kind of nucleic acid, its 3 ' end have can with the part F1c annealed F1 district on same the chain, and the annealing by described F1 district and F1c can form to have comprised and can carry out the F2c district of base pairing at interior ring;

B) with F1c annealed F1 3 ' end be the synthetic complementary strand of starting point;

C) make at its 3 ' end and comprise with the annealing of the oligonucleotide of F2c district complementary F2 sequence and be to synthesize starting point with this oligonucleotide, carry out complementary strand by polysaccharase catalysis strand displacement type complementary strand synthesis reaction and synthesize, with displacement step b) middle institute synthetic complementary strand;

D) make a kind of polynucleotide annealing and be to synthesize starting point with its 3 ' end, carrying out complementary strand by polysaccharase catalysis strand displacement type complementary strand synthesis reaction synthesizes, with displacement step c) in institute synthetic complementary strand, wherein said polynucleotide comprise the arbitrary region complementary sequence of synthetic complementary strand in a kind of and the step c) at its 3 ' end;

(2) as (1) described method, wherein synthetic starting point described in the step d) be on the same chain 3 ' terminal can with R1c district annealed R1 district, the annealing by R1 and R1c can form to have comprised can carry out the R2c district of base pairing at interior ring.

(3) a kind of oligonucleotide, it is made of following two regional X2 and X1c at least, and X1c is connected to 5 ' side of X2,

X2 district: have and contain in the nucleic acid of specific nucleotide sequence X2c district complementary nucleotide sequence arbitrarily;

X1c district: have and the essentially identical nucleotide sequence in X1c district that contains X2c district 5 ' side in the nucleic acid of specific nucleotide sequence.

(4) as (1) described method, wherein the described nucleic acid of step a) is second nucleic acid that produces by following steps:

I) make as the F2 district of oligonucleotide as described in (3) with anneal as the F2c district in the nucleic acid of template, in the wherein said oligonucleotide, the X2 district is the F2 district, the X1c district is the F1c district;

Be synthetic first nucleic acid that has with the nucleotide sequence of described template complementation of starting point ii) with the F2 in the described oligonucleotide;

Iii) make step I i) in any district of synthetic first nucleic acid be in the state that can carry out base pairing;

Iv) make the annealing of a kind of oligonucleotide and be to synthesize starting point with this oligonucleotide, synthetic second nucleic acid, and the F1 that makes this nucleic acid 3 ' end is in the state that can carry out base pairing, wherein said oligonucleotide have with first nucleic acid of step I in ii) in can carry out the nucleotide sequence of the regional complementarity of base pairing.

(5) as (4) described method, wherein can to carry out the zone of base pairing in ii) be R2c to step I, and the oligonucleotide of step I in v) be as (3) described oligonucleotide, and wherein the X2c district is the R2c district, and the X1c district is the R1c district.

(6) as (4 or 5) described method, the state that can carry out base pairing during wherein step I ii) reaches iv) produces by polysaccharase catalysis strand displacement type complementary strand synthesis reaction, the synthetic starting point of this reaction is enabled two kinds of outer primers: a kind of be with template in 3 ' side annealed outer primer of F2c, another kind be with as step I v) in 3 ' side annealed outer primer in zone of the synthetic starting point of first nucleic acid.

(7) as (6) described method, the relation of melting temperature(Tm) below existing under the identical rigorous condition in the described reaction between used every kind of oligonucleotide and its complementation district in template: (outer primer/template 3 ' lateral areas)≤(F2c/F2 and R2c/R2)≤(F1c/F1 and R1c/R1).

(8) as each described method in (4)-(7), wherein said nucleic acid as template is RNA, and step I i) in complementary strand synthesize by enzyme with reverse transcriptase activity.

(9) amplification has the alternately method of the nucleic acid of the complementary nucleotide sequence of connection on one bar chain, and described method is finished by repeating following steps:

A) provide a kind of template, its 3 ' end and 5 ' end are had by being complementary to the district that the nucleotide sequence of each end region is formed on the same chain, when these complementary nucleotide sequences annealing, but form the ring that between carries out base pairing;

B) to be the synthetic complementary strand of synthetic starting point with 3 of the above-mentioned template of same chain annealed ' end;

C) make 3 ' end be complementary to the oligonucleotide and the ring portion annealing of the ring inner nucleotide sequence of 3 ' end side, and be synthetic starting point with this oligonucleotide, synthesize complementary strand by polysaccharase catalysis strand displacement type complementary strand synthesis reaction, with displacement step B) middle synthetic complementary strand, make its 3 ' end be in the state that can carry out base pairing;

D) make step C) in have the chain that can carry out 3 of base pairing ' end as the new template in (A).

(10) as (9) described amplification method, wherein step C) in 5 ' end of oligonucleotide have one section with as step B) the 3 terminal complementary nucleotide sequences that synthesize starting point.

(11) as (10) described amplification method, further comprise such step: with step C) in oligonucleotide be used as steps A as synthetic starting point synthetic complementary strand) in template.

(12) as (9) described amplification method, wherein steps A) template be by method synthetic as described in (5).

(13) as (1) or (9) described method, wherein strand displacement type complementary strand synthesis reaction is to implement in the presence of the melting temperature(Tm) conditioning agent.

(14) as (13) described method, wherein the melting temperature(Tm) conditioning agent is a trimethyl-glycine.

(15) as (14) described method, the concentration that wherein allows trimethyl-glycine in the reaction soln is 0.2-3.0M.

(16) method of target nucleotide sequences in a kind of test sample comprises implementing whether to generate amplified production as each described amplification method in (9)-(15) and observation.

(17), wherein in amplified production, add the probe that comprises with described ring complementary nucleotide sequence, and then observe hybridization between the two as (16) described method.

(18) as (17) described method, wherein said probe is marked on the particle, and observes the aggreation that takes place by hybridization.

(19), wherein in the presence of detection of nucleic acids reagent, implement, and change to observe whether generate amplified production according to the signal of this detection reagent as each described amplification method in (9)-(15) as (16) described method.

(20) with as (16) as described in method detect the method for the sudden change in the target nucleotide sequences, wherein hindered synthesizing of arbitrary complementary strand of forming this amplification method as the sudden change of amplification object in the nucleotide sequence.

(21) synthesize the test kit of the nucleic acid of the complementary nucleotide sequence that has alternately connection on one bar chain, it comprises following component:

I) as (3) described oligonucleotide, be X2c wherein as F2c zone in the nucleic acid of template, the F1c that is positioned at F2c 5 ' end is X1c;

Ii) a kind of oligonucleotide, its comprise be complementary to i) oligonucleotide be primer and the nucleotide sequence of arbitrary region in the synthetic complementary strand;

Iii) a kind of oligonucleotide, its have with as the F3c district complementary nucleotide sequence of F2c district 3 ' side in the nucleic acid of template;

Iv) a kind of archaeal dna polymerase that is used for catalysis strand displacement type complementary strand synthesis reaction and,

V) a kind of Nucleotide, it is as component substrate iv).

(22) as (21) described test kit, wherein oligonucleotide ii) is as (3) described oligonucleotide, with i) oligonucleotide in the synthetic starting point synthetic complementary strand arbitrarily the R2c district be X2c, the R1c that is positioned at R2c 5 ' side is X1c.

(23) as (22) described test kit, further comprise following component:

Vi) a kind of oligonucleotide, it has and use i) oligonucleotide as the R3c district complementary nucleotide sequence of any R2c district 3 ' side in the starting point synthetic complementary strand.

(24) a kind of test kit that is used to detect target nucleotide sequences wherein as described in each on basis of test kit, also further comprises the detection reagent that is used to detect nucleic acid building-up reactions product in as (21)-(23).

Having the nucleic acid that complementary nucleotide sequence alternately is connected in the strand is synthetic purpose of the present invention, and this nucleic acid refers to have mutual complementary nucleotide sequence and be connected nucleic acid in the strand side by side.In addition, it should comprise the nucleotide sequence that is used for Cheng Huan between complementary strand among the present invention.This sequence is called into the ring sequence among the present invention.Synthetic nucleic acid of the present invention is basically by forming by the mutual complementary chain that becomes to encircle the sequence connection.Generally speaking, no matter whether part relates to base pairing, the chain that can not be separated into two or more molecules when the pairing base is separated is called strand.Complementary nucleotide sequence can form base pairing in the same chain.The present invention can obtain the product of intramolecularly base pairing by allowing that having nucleotide sequence alternately is connected nucleic acid base pairing in same chain in the strand, and this product is supplied with the ring of forming obviously double-stranded district and not relating to base pairing.

Just, the present invention has the nucleic acid that complementary nucleotide sequence alternately is connected in the strand can be defined as single-chain nucleic acid, wherein comprise can be in same chain the annealed complementary nucleotide sequence, and its annealing product is partly formed the ring that does not relate to base pairing at flexure hinge.Nucleotide with complementary nucleotide sequence can be annealed into the ring that does not relate to base pairing.Becoming the ring sequence can be nucleotide sequence arbitrarily.Become the ring sequence base pairing to be used for the synthetic of metathetical complementary strand with startup.And preferentially be provided the sequence different with the nucleotide sequence that is positioned at other district, to obtain specificity annealing.For example, in an embodiment preferred, become the ring sequence to comprise the identical nucleotide sequence with F2c (or R2c) district basically, F2c (or R2c) district is positioned at the 3 '-side in the district (for example F1c or R1c) that comes from template nucleic acid, and anneals in same chain.

Essentially identical nucleotide sequence is defined as follows among the present invention.Just, when with certain sequence as the annealing of template synthetic complementary strand and target nucleotide sequences when supplying with the starting point of synthetic complementary strand, this certain sequence is identical with target nucleotide sequences basically.For example, identical with F2 basically sequence not only comprises the sequence identical with F2 fully, and also comprising can be as the nucleotide sequence of template, and described template can provide the starting point with F2 annealed nucleotide sequence and the synthetic complementary strand of energy conduct.Term of the present invention " annealing " refers to by the base pairing according to Wo Sen-Ke Like law, forms the nucleic acid of duplex structure.Therefore, be strand even form the nucleic acid chains of base pairing, if the base pairing of intramolecularly complementary nucleotide sequence, annealing also can take place.Since form duplex structure by base pairing nucleic acid, so the present invention's annealing has the identical meaning with hybridization.

The nucleotide sequence logarithm that the present invention forms nucleic acid is at least 1.The model that the present invention is desired, the nucleotide sequence logarithm can be 1 integral multiple.In this situation, the complementary nucleotide sequence logarithm that the present invention forms Nucleotide does not have the upper limit in theory, and when the synthetic product nucleic acid of the present invention that is made of many groups complementary nucleotide sequence, this nucleic acid is formed by repeating identical nucleotide sequence.

Synthetic of the present invention has the nucleic acid that complementary nucleotide sequence alternately is connected in the strand can not have identical structure with naturally occurring nucleic acid.If known when by nucleic acid polymerase effect nucleic acid with nucleotide derivative as substrate, but nucleic acid derivative just.Used nucleotide derivative comprises the nucleotide derivative biological example element or the digoxin of the Nucleotide or the binding partner mark of labelled with radioisotope.These nucleotide derivatives can be used for marked product nucleic acid.Perhaps, if substrate is a fluorescent nucleotide, then product nucleic acid may be fluorescent derivative.And product can be DNA and also can be RNA.The product that generates is by in conjunction with the primer structure that realizes the nucleic acid polymerization reaction, polyreaction substrate type, the reagent of polyreaction and deciding.

The nucleic acid that utilizes archaeal dna polymerase to start said structure is arranged synthetic, but this archaeal dna polymerase have strand displacement activity and F1 district possess 3 '-formation of annealing of part F1c district on terminal and the same chain comprises the F2c district of base pairing at interior ring.Many reports about complementary strand synthesis reaction are arranged, wherein form hairpin loop, from as template, and offer the district that the hairpin loop part can base pairing among the present invention, and have the new feature that when synthetic complementary strand, utilizes this district with the hairpin loop sequence.By distinguishing, before be replaced as template synthetic complementary strand certainly with the hairpin loop sequence as the synthetic starting point.Then be positioned at the state of replacing chain 3 '-terminal R1c district (district arbitrarily) but being in a kind of base pairing.Have that to carry out complementary strand by annealing synthetic with the district of this R1c complementary sequence, cause generating nucleic acid (2 molecule), this nucleic acid has nucleotide sequence and its complementary strand of extending to R1c from F1 and forms by being bonded to each other by becoming to encircle sequence.Can at random select for example top R1c in district arbitrarily among the present invention, if it can distinguish the polynucleotide annealing of complementary nucleotide sequence with this.And be synthetic starting point synthetic complementary strand with polynucleotide, this synthetic complementary strand has requisite effect to the present invention.

The present invention uses term " nucleic acid ", and nucleic acid of the present invention not only comprises DNA but also comprise RNA usually.Yet function is for the template of synthetic complementary strand, and nucleic acid or the modified nucleotide replaced by artificial derivative from its Nucleotide of n DNA or RNA are also included within the nucleic acid scope of the present invention.Common nucleic acid of the present invention is contained in the biological sample, and biological sample comprises animal, the tissue of plant or microorganism, cell, culture and secretory product, or their extract.Biological sample of the present invention comprises cytozoon genomic dna or RNA for example virus or mycoplasma.Nucleic acid of the present invention is generally derived by the nucleic acid that is included in described biological sample.For example by the synthetic cDNA of mRNA, derive the nucleic acid that comes and the nucleic acid that increases based on biological sample, be the representative instance of nucleic acid of the present invention.

The feature of nucleic acid of the present invention is to be provided the F1 district at 3 '-end, can with part F1c on same chain annealing, anneal by the F1c on this F1 district and the same chain, but can form comprise base pairing the F2c district at interior ring, can in the whole bag of tricks, obtain this nucleic acid.In the most preferred embodiment, utilize the oligonucleotide that following structure is arranged, can provide this structure by the reaction of synthesizing complementary strand.

Just, the effective oligonucleotide of the present invention is made of following two district X2 and X1c at least, and wherein X1c links to each other with 5 ' of X2-side.

The X2 district: have with nucleic acid in X2c district complementary nucleotide sequence, described nucleic acid has specific nucleotide sequence.

The X1c district: the substantially the same nucleotide sequence with the X1c district is arranged, and described X1c district is positioned at X2c district 5 '-side in the nucleic acid with specific nucleotide sequence.

Refer to by the nucleic acid that oligonucleotide structure of the present invention determined herein and serve as the nucleic acid of template when being primer with oligonucleotide of the present invention with specific nucleotide sequence.Detecting in the nucleic acid situation based on synthetic method of the present invention, described nucleic acid with specific nucleotide sequence is the target that detects or detects target deutero-nucleic acid.Nucleic acid with specific nucleotide sequence refers to wherein, and at least a portion nucleotide sequence is disclosed or predictable.Disclosed partial nucleotide sequence is the X2c district and is positioned at its 5 '-side X1c district.Can infer that these 2 districts are contiguous or separate existence.By the relative position in two districts, in the time of can be by product nucleic acid self-annealing, the ring portion state of formation decides.The distance in preferred two districts is disconnected from each other not far so that make the nucleic acid product self-annealing have precedence over intermolecular annealing.Therefore, two zone position concern preferably contiguous, and distance is generally 0 to 500 base.Yet, at following self-annealing Cheng Huanzhong, such situation being arranged, the mutually too near state in described two districts of expectation will be unfavorable for the formation that encircles down.In ring, need a kind of annealing oligonucleotide and be the structure that the synthetic starting point starts the complementary strand reaction of strand displacement smoothly of having recently been widowed with described oligonucleotide.More preferably, X2c district and the distance that is positioned at its 5 '-side X1c district are designed to 0 to 100 base, more are contemplated to be 10 to 70 bases.Numerical value is not for comprising the length of X1c and X2.The base of formation loop section is counted this length and is added the district that is equivalent to X2.

Composition based on the used term of nucleotide sequence feature " identical " of oligonucleotide of the present invention with " complementary " and do not mean that absolute identical and absolute complementation.Just, identical with certain sequence sequence comprises and certain sequence annealed nucleotide sequence complementary sequence.On the other hand, complementary sequence is an energy annealed sequence under the stringent condition, provides as complementary strand synthetic starting point 3 '-end.

Usually, for the nucleic acid with specific nucleotide sequence, the X2 district of composition oligonucleotide of the present invention and X1c zone position are close to and are not had overlapping.If common ground is arranged in the nucleotide sequence, two districts just have part and cover, because X2 plays the function of primer, it is 3 '-end always.On the other hand, X1c as described below supplies with complementary strand 3 '-end with the effect of primer, and this complementary strand is that template is synthetic by nucleic acid, therefore should be designed in 5 '-end.With this oligonucleotide is that the synthetic starting point obtains complementary strand, is the synthetic reverse complemental chain of template with this complementary strand in next step.And final oligonucleotide of the present invention partly is the template into complementary strand that is copied.3 '-end that copy generates has nucleotide sequence X1, and this sequence is annealed into ring with X1c in same chain.

Among the present invention, oligonucleotide is the Nucleotide that satisfies two requirements, promptly must can form the complementary base pairing, and is complementary strand synthetic starting point at 3 '-end supply-OH base.Therefore, its main chain and be not necessarily limited to a kind of connection of phosphodiester bond.For example, the peptide nucleic acid(PNA) that it can be formed main chain or be connected based on peptide by the thiophosphoric acid derivative, described thiophosphoric acid derivative is that S replaces O.But base is meant the base of those complementary pairings.Five kinds of bases of natural existence, i.e. A, C, T, G and U, base also can be for example bromodeoxyribouridine of analogue.Preferably, oligonucleotide of the present invention not only can be used as the synthetic starting point and also can be complementary strand synthetic template.Term polynucleotide of the present invention comprise oligonucleotide.The chain length of term used herein " polynucleotide " is not limited, and used term " oligonucleotide " refers to the nucleotide polymer of short relatively chain length.

Under given condition, oligonucleotide chain of the present invention has and can and keep certain a kind of like this length of specificity with the complementary strand base pairing in the following various nucleic acid building-up reactions.Particularly, it is by 5-200 based composition, more preferably 10-50 base pair.The chain length of discerning known polysaccharase is at least 5 bases.This polysaccharase catalysis relies on the nucleic acid building-up reactions of sequence.So the chain length of annealing portion should be longer than this length.In addition, the length of desired 10 bases or longer on the statistics to obtain desired Nucleotide specificity.On the other hand, because chemosynthesis prepares relatively difficulty of oversize nucleotide sequence.Therefore above-mentioned chain length is the example of desired scope.The chain length of illustration refers to part and complementary strand annealed chain length.As following described, oligonucleotide of the present invention can finally be annealed with two districts at least respectively.Therefore, the chain length of illustration is interpreted as forming the chain length in each district of oligonucleotide here.

In addition, oligonucleotide of the present invention can be with known marker mark.Marker comprises binding partner for example digoxin and vitamin H, enzyme, fluorescence, shiner, radio isotope.Well-known by the fluorescence analogue replace the base of forming oligonucleotide technology (WO95/05391, Proc.Natl.Acad.Sci.USA, 91, 6644-6648,1994).

Other oligonucleotide of the present invention also can be incorporated into solid phase.Perhaps, the arbitrary portion of oligonucleotide can be used the binding partner mark, the biological example element, indirectly by binding partner for example fixedly avidin fixed.When immobilized oligonucleotide was the synthetic starting point, building-up reactions product nucleic acid was caught by solid phase, helps its separation.Can detect separate part by the nucleic acid specificity indicator or with the hybridization of label probe.Target nucleic acid fragment also can be reclaimed by any restriction enzyme digestion product.

Term used herein " template " is meant when being used for synthetic complementary strand the nucleic acid as template.Have nucleotide sequence and the template complementary complementary strand meaning and be meant chain corresponding to template.But the relation of the two is relative.It is the function that the synthetic complementary strand can play template once more.Just, complementary strand can be used as template.

The useful oligonucleotide of the present invention is not limited to above-mentioned 2 districts, can comprise further region.X2 and X1c are designed into 3 ' respectively-and 5 '-end, arbitrary sequence can be inserted betwixt.Its recognition sequence of restriction enzyme for example, the promotor that RNA polymerase is discerned, or the DNA of encoding ribozyme.By utilizing its recognition sequence as restriction enzyme, synthetic of the present invention has the complementary sequence and alternately is connected to the double chain nucleotide that nucleic acid in the strand can be fragmented into same length.Promoter sequence is designed to RNA polymerase can discern, synthetic product of the present invention as template to allow further to be transcribed into RNA.Also can be by the DNA of design encoding ribozyme, transcription product just can be realized from the system of fracture.These extra nucleotide sequences are that those are forming the sequence that plays a role after the two strands.Therefore, when strand Nucleotide of the present invention became ring, these sequences were inoperative, just play a role when nucleic acid is extended and be annealed into the chain with complementary nucleotide sequence under the missing link situation.

When promotor when combining with the direction that allows synthetic district to transcribe based on oligonucleotide of the present invention, the reaction product of the present invention at the position that identical nucleotide sequence is repeated realizes efficient re-reading system.By this system is combined with appropriate expression system, just can translate albumen.Promptly utilize this system at bacterium or zooblast or in in-vitro transcription with translate into albumen.

But chemosynthesis the present invention has the oligonucleotide of said structure.Perhaps, natural acid can change forming of above-mentioned base sequence or their mode of connection by for example restriction enzyme fracture.

The ultimate principle of the invention process building-up reactions is with reference to figure 5-Fig. 6, and this reaction is implemented by utilizing above-mentioned useful oligonucleotide to combine with archaeal dna polymerase, and this archaeal dna polymerase has the activity of strand replacement reaction in the nucleic acid building-up reactions.At first X2 (corresponding to F2) is as template, and above-mentioned oligonucleotide (FA among Fig. 5) and template nucleic acid annealing are to provide complementary strand synthetic starting point.Replaced by outer primer (F3) synthetic complementary strand (following) by the synthetic starting point synthetic complementary strand of FA among Fig. 5 and formed strand (Fig. 5-A).When further synthesizing complementary strand, this complementary strand and the complementary strand complementation that is just generating, Fig. 5-A synthetic complementary strand nucleic acid 3 '-end has and oligonucleotide complementary nucleotide sequence of the present invention.Promptly because oligonucleotide 5 ' of the present invention-end has the identical sequence with X1c district (corresponding to F1c).So synthetic nucleic acid 3 '-end has complementary sequence X1 (F1).It is that synthetic starting point synthetic complementary strand is replaced by synthetic starting point synthetic complementary strand by outer primer R3 that Fig. 5 shows from R1.In case but 3 '-terminal portions makes its base pairing by this replacement, the 3 '-terminal X1 (F1) on same chain and X1c (F1c) annealing is carried out with from as the extension of template (Fig. 5-B).Then, be positioned at its 3 '-terminal X2c (F2c) and form ring, this ring does not relate to base pairing.X2 in the oligonucleotide of the present invention (F2) is annealed into this ring, is the synthetic complementary strand of synthetic starting point (Fig. 5-B) with described oligonucleotide.With the first synthetic product product that is the building-up reactions of template, be replaced by strand replacement reaction and make its base pairing.

The present invention be by can carrying out any reverse primer of nucleic acid synthetic with a kind of basic composition is, and wherein said nucleic acid is to be that primer synthetic complementary strand is as template with this oligonucleotide.Most of nucleic acid synthetic products can be obtained as shown in Figure 6.As can be seen from Figure 6, be the desired nucleic acid product of the present invention (D), have the complementary nucleotide sequence and alternately be connected in the strand.In a single day thermally denature is transformed into strand by for example handling, and other product (E) is once more as the template that forms (D).If double-stranded product (D) nucleic acid is converted into strand by thermally denature, generation is annealed high probability and can not be formed initial two strands in the same chain.This is to have precedence over intermolecular reaction because of the intramolecular reaction with complementary strand of identical melting temperature(Tm) (Tm) to carry out.Each the bar strand of product (D) deutero-of annealing from same chain is annealed in same chain, and turns back to (B) state.Every chain is further supplied with (D) molecule and (E) molecule respectively.By repeating these steps, might synthesize continuously and have complementary nucleotide sequence and alternately be connected nucleic acid in the strand.The template and the product that form in 1 circulation increase with exponential form, therefore make reaction very effective.

Be realization Fig. 5 (A) state, initial synthetic complementary strand, at least in reverse primer annealed part, but base pairing.This step can be accomplished by any means.At initial template, especially prepared an outer primer, this outer primer is than more being annealed with the F3c district of 3 '-side on template by oligonucleotide annealed F2c of the present invention district.If synthetic by polysaccharase catalysis strand displacement type complementary strand, this outer primer is used as the synthetic starting point with synthetic complementary strand, the present invention is replaced for synthetic starting point synthetic complementary strand from F2c, and the result makes its base pairing (Fig. 5) by R1 and the annealing of R1c district.By utilizing strand replacement reaction, this reaction can be carried out under isothermal condition up to now.

When using outer primer, carry out synthesizing after F2c is synthetic, beginning from outer primer (F3).In the simplest method, make inner primer concentration be higher than the concentration of outer primer.Specifically, the primer is usually at 2 times to 50 times, preferred 4 times to 10 times concentration, and therefore reaction can be by desired carrying out.In addition, the Tm (being equivalent to F1 and R1) that outer primer melting temperature(Tm) (Tm) is lower than X1 in the inner primer is set, therefore can controls the synthetic time.Just, (outer primer F3:F3c)≤F2c/F2)≤(F1c/F1) or (outer primer/in the district of template 3 '-side)≤(X2c:X2)≤(X1c:X1).Here, (F2c/F2)≤(F1c/F1) be to be annealed into ring with F2 because annealing is preferential between F1c/F1, annealing is intramolecular reaction between F1c/F1, and can so high probability preferentially carry out.Yet, consider that in order to provide more reaction conditions Tm is significant.Even in the design reverse primer, what similar reaction conditions was natural should be taken into account.By utilizing this relation, can obtain the condition of statistics coideal reaction.If other condition is constant, by length and the base of forming base pairing in conjunction with the annealed complementary strand, melting temperature(Tm) (Tm) can be calculated in theory.Therefore, those those skilled in the art can derive better condition according to disclosed specification sheets.

In addition, be called the selection of the contiguous phenomenon control outer primer annealing time of piling up.Contiguous pile up make can not independent annealed oligonucleotide can the annealed phenomenon on contiguous double-stranded part (ChiaraBorghesi-Nicoletti et al., Bio Techniques, 12, 474-477 (1992)).Be that outer primer is designed to be close to F2c (X2c) and can not anneal independently.By doing like this, when F2c (X2c) annealed, initial outer primer could be annealed, so the annealing of F2c (X2c) preferentially carries out, and according to this principle, example shows as the nucleotide sequence of the essential oligonucleotide of series reaction primer set.This step also can be achieved by heat denatured or with dna helicase.

If having the template nucleic acid of F2c (X2c) is RNA, Fig. 5-(A) state also can be realized by diverse ways.For example, if this RNA chain is decomposed, but make the R1 base pairing.Just make among F2 and the RNA F2c annealing and be DNA by ThermoScript II synthetic complementary strand.Handle the RNA act in the DNA/RNA two strands by alkaline denaturation or with rnase, be decomposed, wherein form strand from F2 synthetic DNA as the RNA of template.Because enzyme selectivity decomposes the RNA among double-stranded DNA/RNA, some ThermoScript II or the ribonuclease activity of RNase H are used.In this mode, make the R1c annealing of reverse primer and energy base pairing.Therefore, needn't need to cause R1c to be in the outer primer of base pairing.

Perhaps, ThermoScript II strand displacement activity can be used in by the strand displacement as above-mentioned outer primer, in this situation, reactive system can only constitute by ThermoScript II, be template promptly with RNA, might synthesize complementary strand and from being annealed into F3c outer primer F3 for the synthetic complementary strand of synthetic starting point and replace previous synthetic complementary strand simultaneously, outer primer F3 is positioned at 3 ' of F2c-side by ThermoScript II annealing of F2 and F2c in template.When ThermoScript II is template when synthesizing the reaction of complementary strand with DNA, all reactions of synthesizing complementary strand by ThermoScript II comprise with R1c annealed R1 synthetic as the complementary strand of synthetic starting point, this complementary strand in strand replacement reaction as template; Answer to send out as the synthetic and strand displacement simultaneously of the complementary strand of synthetic starting point with R3c annealed R3, R3 is positioned at 3 ' of R3c-side.In institute to can not estimate under the reaction conditions that ThermoScript II shows that the DNA/RNA strand displacement is active the time, can be in conjunction with having as the active archaeal dna polymerase of above-mentioned strand displacement.As the above-mentioned pattern that with RNA is template obtains article one single-chain nucleic acid is preference pattern of the present invention.On the other hand, if use the archaeal dna polymerase that not only has the strand displacement activity but also reverse transcriptase activity arranged such as the Bca archaeal dna polymerase, synthetic from article one single-chain nucleic acid of RNA not only by identical enzyme, and subsequent be that the reaction of template can be carried out similarly with DNA.

There are the reverse primer of ad hoc structure, the above-mentioned reactive mode of the present invention to cause various intrinsic to change by utilization.The most effective variation is described below.Just, the oligonucleotide described in the composition [5] is used as reverse primer in the best model of the present invention.[5] oligonucleotide in is wherein to be a kind of oligonucleotide that any district R2c in the primer synthetic complementary strand and R1c are respectively X2c and X1c with F2.By using such reverse primer, series reaction (forward with reverse) takes place in sense strand and the antisense strand, described series reaction is meant annulation and the reaction that becomes and replace complementary strand from this cyclization.The reaction of effective nucleic acid is greatly improved among the present invention as a result, and this nucleic acid has complementary nucleotide sequence and alternately is connected on the strand, and a series of such reactions can be carried out under the isothermal condition.Hereinafter, this model that Fig. 1 to 3 summarized has more specifically been described as a reference.

In the following surface model, prepared based on 2 kinds of oligonucleotide of the present invention.In order to illustrate, the district that its called after FA and RA. is formed FA and RA is as described below:

X2?X1c

FA F2?F1c

RA R2?R1c

Here, F2 is the complementary nucleotide sequence in F2c district in the template nucleic acid.R2 and any district R2c complementary nucleotide sequence, it is in the primer synthetic complementary strand that R2c is included in F2.F1c and R1c are respectively any nucleotide sequences that is positioned at F2c and R2c downstream.Distance between F2 and R2 arbitrarily.Even the about 1kbp of its length, under the appropriate condition, fully synthetic is feasible, although depend on the synthesis capability of the archaeal dna polymerase that is used to implement synthetic complementary strand, specifically, when using the Bst archaeal dna polymerase, if the distance of F2 and R2c is 800bp, preferred 500bp or littler is with regard to a synthetic surely required product.Relate among the PCR of temperature cycle, by emphasis change temperature enzymic activity is reduced and is considered to reduce longer nucleotide sequence combined coefficient.In the preferred pattern of the present invention, do not need temperature cycle in the amplification of nucleic acid step, therefore in addition the synthetic and amplification of longer nucleotide sequence can be sure of and can realize.

At first, make F2 annealing among the FA at template nucleic acid, and as complementary strand synthetic starting point.Subsequent reactions steps up to Fig. 1 (4) is identical with the previous described key model of the present invention (Fig. 5).Anneal sequence is F3 among Fig. 1 (2), is exactly above-mentioned outside primer.The archaeal dna polymerase that carries out strand displacement type complementary strand synthesis reaction in order to this primer for synthetic starting point is replaced and is in base pairing from FA synthetic complementary strand.

When R2c is in base pairing in (4), annealing in this of R2c/R2 in conjunction with lining reverse primer RA.Carry out the synthetic F1c that arrives at FA5 '-end up to this chain extension of complementary strand for synthetic starting point with this position.After the reaction of this synthetic complementary strand, the outer primer R3 that is used to replace is at the synthetic complementary strand of this annealing, and strand displacement is also carrying out during this period, makes from RA to be replaced for synthetic starting point synthetic complementary strand.In the complementary strand of Ti Huaning, RA is positioned at its 5 '-end thus, is positioned at its 3 '-end with FA complementary sequence.

Therefore in the single-chain nucleic acid 3 ' that is replaced-side, have sequence on the same chain with F1c complementary F1.F1 anneals to start the synthetic of complementary strand with the F1c that same intramolecularly is arranged rapidly.When 3 '-end (F1) in same chain is annealed with F1c, form the ring that contains F2c.Clearly show as Fig. 2-(7), but this loop section base pairing.The annealing of oligonucleotide FA of the present invention and this loop section, and as the synthetic starting point of complementary strand (7), described oligonucleotide has and F2c complementary sequence.When the reaction product when the complementary strand that formerly starts from F1 is synthetic is replaced, carry out the synthetic of complementary strand from ring.But the result from as template synthetic complementary strand in the base pairing once more of 3 '-end.This 3 '-end is provided the R1 district that can be annealed to R1c in the same chain, and because intramolecular being swift in response, described two chains are preferentially annealed.With above-mentioned reacting phase with from being that the reaction that template synthetic 3 '-end begins is also carried out in this district with FA.As a result, by synthetic complementary strand of successive and subsequent displacement, the present invention has the nucleic acid that complementary nucleotide sequence alternately is connected to identical strand, is that starting point is continued to extend from 3 '-terminal R1.Because R2c always is comprised in the ring that the intramolecularly annealing by 3 '-terminal R1 forms, and provides the oligonucleotide (RA) of R2 to be annealed into ring at 3 '-end in subsequent reaction.

When attention concentrates on when oligonucleotide begins to be synthesized into complementary strand nucleic acid, this nucleic acid is to be extended as template certainly, described oligonucleotide is annealed into ring in single-chain nucleic acid, the present invention has complementary nucleotide sequence and alternately is connected the synthetic of nucleic acid in the strand and also carrying out.Promptly when from the complementary strand of ring be synthesized in Fig. 2-(7) RA the time, synthetic being accomplished.Then, when the synthetic startup complementary strand (Fig. 3-(8)) of the nucleic acid of replacing by this nucleic acid was synthetic, reaction arrived the ring of the synthetic starting point of Ceng Zuowei, and replaces and restart.In this mode, the nucleic acid that begins to be synthesized from ring also is replaced.The result obtains can annealed 3 '-terminal R1 (Fig. 3-(10)) in the same chain.This 3 '-terminal R1 anneals to start the synthetic of complementary strand with R1c in same chain.Except substitute with F the R should reaction with Fig. 2-(7) in identical.Therefore, structure shown in Fig. 3-(10) has the effect of playing new nucleic acid to be continued oneself's extension and forms new nucleic acid.

Opposite with above-mentioned reaction, reaction from the nucleic acid promoter nucleic acid shown in Fig. 3-(10), cause beginning to extend, that is to say that a nucleic acid is extended among the present invention from synthetic starting point 3 '-terminal F1, continue to provide the reaction that can start the new nucleic acid that extends to carry out respectively, and, along with the extension of chain, not only produce many one-tenth ring sequences endways but also on same chain, when but these became the ring sequence through the strand replacement reaction base pairing, the starting point of the reaction that forms new nucleic acid was served as in oligonucleotide annealing.By not only endways but also the building-up reactions that in chain, begins, obtain further effectively amplification.Be combined into aforesaid reverse primer based on oligonucleotide RA of the present invention, wherein extend and carried out with the formation of new nucleic acid subsequently.And, should obtain extension by the new nucleic acid that forms itself among the present invention, also caused the formation of back new nucleic acid, a series of such reactions can for good and all obtain very effective nucleic acid amplification in theory.In addition, reaction of the present invention can be carried out under isothermal condition.

Therefore, the reaction product of accumulation has a kind of structure, this structure has nucleotide sequence between F1 and R1, and its complementary strand alternately connects, yet repeating unit two ends all have the district that is made up of continuous nucleotide sequence F2-F1 (F2c-F1c) or R2-R1 (R2c-R1c), for example, in Fig. 3-(9), sequence (R2-F2c)-(F1-R2c)-(R1-F1c)-(F2-R2c) connect in this order from 5 '-side.This is because carry out with a kind of like this principle based on amplified reaction of the present invention, promptly react with oligonucleotide as synthetic starting point from F2 (or R2), then, complementary strand is by being extended with self 3 '-terminal reaction from F1 (or R1) synthetic as synthetic starting point.

Here, in most preferred model, oligonucleotide FA of the present invention and RA are the oligonucleotide that is annealed into loop section.Even yet do not use these to have the oligonucleotide of limiting structure, amplified reaction of the present invention can be achieved by utilizing the complementary strand synthetic oligonucleotide that can start from the ring beginning, just, the 3 '-end that is extending, in case replaced since ring beginning synthetic complementary strand, supplied with loop section with regard to another.Because from the complementary strand of ring beginning is synthetic, always alternately be connected in the strand nucleic acid as template to have complementary nucleotide sequence.Clearly, can synthesize nucleic acid required for the present invention.Yet so synthetic nucleic acid carries out the synthetic of complementary strand by replacing back formation ring.But therefore the 3 '-end that not can be used for subsequent formation ring can not use as new template.Do not start synthetic nucleic acid so do not resemble by FA or RA, the product in this situation can not obtain desirable exponential amplification.Owing to this reason, the oligonucleotide with FA or RA structure can be used for synthetic efficiently nucleic acid of the present invention.

A series of such reactions are undertaken by following description, composition below the template single-chain nucleic acid adds, and then can form at the nucleotide sequence of composition FA and RA and its complementary nucleotide sequence and stablize base pairing and culturing mixt under a kind of like this temperature that the activity of enzyme can keep.

4 kinds of oligonucleotide:

FA，

RA，

Outer primer F3 and

Outer primer R3,

Be used to carry out displaced type complementary strand synthetic archaeal dna polymerase,

Oligonucleotide as the archaeal dna polymerase substrate.

Therefore, need not to set the temperature cycle of PCR and so on.Stable base pairing refers to the state that at least a portion oligonucleotide in the reactive system can provide the synthetic starting point of complementary strand here.For example, can cause stablizing the required condition temperature of base pairing and be set to be lower than melting temperature(Tm) (Tm).Generally speaking, melting temperature(Tm) (Tm) is that the nucleic acid with complementary nucleotide sequence has 50% base pairing.Among the present invention temperature being set at melting temperature(Tm) (Tm) or being lower than melting temperature(Tm) is not most important condition, but is considered to reach reaction conditions of efficient synthetic.If template used nucleic acid is double-stranded, but described nucleic acid at least in the oligonucleotide annealed zone should base pairing.For this reason, generally carry out thermally denature, and be that pre-treatment is only carried out once at reaction beginning previous crops.

This is reflected at following composition and exists down and carry out, and can make enzyme reaction be in the damping fluid of suitable pH value, anneals or keeps the essential salt of enzymatic activity, the medium of protective enzyme and regulate and control the necessary modulator of melting temperature(Tm) (Tm).For damping fluid, for example used Tris-HCl that shock absorption is arranged in neutrality or weakly alkaline scope.Regulate the pH value according to used archaeal dna polymerase, for salt, KCl, NaCl, (NH ₄) ₂SO ₄Add with activity that keeps enzyme and the melting temperature(Tm) (Tm) of regulating and control nucleic acid Deng an amount of, the medium of protective enzyme uses bovine serum albumin or carbohydrate.In addition, generally use dimethyl sulfoxide (DMSO) (DMSO) or methane amide modulator as melting temperature(Tm) (Tm).The annealing of the oligonucleotide under the temperature condition that limits of the modulator by utilizing melting temperature(Tm) (Tm) has obtained regulation and control.And, trimethyl-glycine (N, N, N-Trimethyl glycine) or tetraalkylammonium salt (tetraalkyl) by its etc. stabilization (isostabilization) also be effective for the efficient of improving strand displacement.By add the 0.2-3.0M trimethyl-glycine in reaction soln, preferred 0.5-1.5M can obtain the promoter action of desirable the present invention to nucleic acid amplification.Because the modulator of these melting temperature(Tm)s has the effect that reduces melting temperature(Tm), preciseness that those are suitable and reactive conditions will be in conjunction with the concentration of salt, and temperature of reaction etc. are decided by rule of thumb.

Kept unless the important feature of the present invention is the position relation in many districts, otherwise series reaction can not be carried out.Because this feature follows the non-special building-up reactions of the non-special synthetic of complementary strand to obtain effective prevention.Even certain non-specific responding just takes place, product has also obtained reduction as the possibility of initial substance in synthetic subsequent amplification step, and, by the progress of many districts regulation and control reaction, the detection system that might cause identifying required product accurately in similar nucleotide sequence can optionally be formed.

Utilize described feature detection transgenation.The present invention uses in the outer primer pattern, used 4 primers, i.e. 2 primers of 2 outer primers and oligonucleotide of the present invention composition.If described 6 districts are included in inoperative words in 4 oligonucleotide, building-up reactions then of the present invention can not be carried out.Particularly, be important sequence as the sequence of each oligonucleotide 3 '-end of the synthetic starting point of complementary strand with described complementary strand for the sequence of sequence X 1c district 5 '-end of synthesizing starting point.Therefore design these important sequences and make corresponding sudden change energy detected, the product of building-up reactions of the present invention can be observed, and exists or does not exist such as the sudden change of base deletion or insertion or such as the genetic polymorphism of SNPs and can be analyzed widely.More specifically, design estimate at sudden change or the base of polymorphism make its with as near oligonucleotide 3 '-end of the synthetic starting point of complementary strand or suitable at complementary strand as the base of 5 ' of synthetic starting point-end, mismatch if exist near 3 ' of the synthetic starting point of complementary strand-terminal or its, the reaction of complementary strand nucleic acid is subjected to suppressing significantly.Unless among the present invention in the initial reaction structure of product end cause reaction repeated, otherwise can not obtain highly the reaction of amplification.Therefore, even, constitute always being interrupted synthesizing in certain step of complementary strand of amplified reaction, therefore, under situation about mismatching, can not have the reaction of highly increasing making a mistake when synthetic.The result mismatches to have suppressed amplified reaction effectively, finally causes producing result accurately.Just we can say based on the amplified reaction of nucleic acid of the present invention the inspection of nucleotide sequence is had a highly complete mechanism.These features are the advantages that only are difficult to reckon with in the PCR method that 2 districts carry out such as amplified reaction.

Behind the synthetic complementary sequence, show that the X1c district of the used oligonucleotide feature of the present invention can be used as the synthetic starting point, and X1 sequence annealing in this complementary sequence and the new same chain of synthetic, building-up reactions wherein is to carry out as template certainly.Therefore, even be formed on the so-called primer dipolymer that usually becomes problem in the area research of forward position, this oligonucleotide can not Cheng Huan.Therefore, the non-specific amplification owing to the primer dipolymer forms can not take place in theory.Thus, described oligonucleotide helps to improve the specificity of this reaction.

In addition, outer primer F3 shown in the present (Fig. 1-(2)) or R3 (Fig. 2-(5)) are combined and above-mentioned series reaction is carried out under isothermal condition.Just, the invention provides a kind of amplification and have the method that complementary sequence alternately is connected the nucleic acid in the strand.Wherein be included in the step shown in above-mentioned 9.In this method, between F2c/F2, between R2c/R2, between F1c/F1 and select to take place the temperature condition of stabilizing annealing between R1c/R1, by promoting contiguous (contiguous stacking) phenomenon of piling up preferably to determine between F3c/F3 and R3c/R3, to anneal respectively in the annealing of F2c/F2 and R2c/R2.

The present invention uses that term nucleic acid " synthesizes " and " amplification ".The synthetic nucleic acid that begins from the oligonucleotide as synthetic starting point that refers to of nucleic acid of the present invention extends.If the extension of the reaction of not only described synthetic but also the formation of other nucleic acid and the nucleic acid that forms can be carried out continuously, a series of such reactions broadly are called amplification.

The single-chain nucleic acid that is produced 3 '-end have can with F1c part annealed F1 district in the same chain, carry out the annealing in F1 district and F1c in the same chain, but can form the ring in the F2c district of containing base pairing, be the important composition of the present invention.Such single-chain nucleic acid can also following principle mode supply with.Just, the primer of the synthetic basis of complementary strand with following structure just allowed and carried out.5 '-[with annealed X1 district in X1c district in the primer]-[can form base pairing sequence ring]-[X1c district]-[having and template complementary sequence area]-3 '.

Preparation has and template complementary sequence area, two nucleotide sequences, i.e. a nucleotide sequence (primers F A) complementary and a nucleotide sequence (primer RA) and R1c complementation with F1.Form that the nucleotide sequence composition want nucleic acid comprises the nucleotide sequence that extends to the R1c district from the F1 district and from having the nucleotide sequence that extends to the F1c district with the R1 district of this nucleotide sequence complementary nucleotide sequence.Can be in primer annealed X1c and X1 can be sequence arbitrarily.Yet, in the district between primers F A and RA, preferably make the sequence difference in this X1c/X1 district.

At first, carry out the synthetic of complementary strand from template nucleic acid F1 district by primers F A, but then R1c district base pairing in synthetic complementary strand, make other primer annealing form the synthetic starting point of complementary strand, synthetic complementary strand 3 ' in this step-end by with the primers F A complementary sequence of forming initial synthetic chain 5 '-end.Therefore be provided the X1 district at its 3 '-end, wherein the X1 district is annealed into ring with the X1c district in same chain.The structure of characteristic 3 ' of the present invention-end is provided thus, and the system that before had been shown as most preferred mode is formed in subsequent reaction.The oligonucleotide that is annealed into loop section is provided the X2 district at 3 '-end, is provided the X1 district at 5 '-end, and the X2c district is complementary in X2 district and the ring.In the reactive system formerly, synthesize and template nucleic acid complementary chain, ring structure is supplied with 3 ' of nucleic acid-end with primers F A and RA.In this method, just can provide characteristic end structure of the present invention effectively by very short primer.On the other hand, in the described model, the complete nucleotide sequence of makeup ring can be used as primer and is provided, and must synthetic long primer.

Can be used as reverse primer if comprise the nucleotide sequence of restriction enzyme cog region, then can make up the different model of the present invention.The reaction that utilization comprises the reverse primer of restriction enzyme recognition sequence specifically describes with reference to figure 6.As Fig. 6-when (D) being accomplished, produce otch by restriction enzyme and be equivalent to restriction enzyme recognition site in the reverse primer.Begin the reaction of synthetic strand displacement type complementary strand with this otch for synthetic starting point.Because reverse primer is positioned at two ends of the double-strandednucleic acid of composition (D), the reaction of synthetic complementary strand also can be terminal since two, although basically based on the SDA method described in the tip technology, the present invention has the structure that complementary nucleotide sequence alternately connects as the nucleotide sequence of template, thereby constitutes the unique nucleic acid of the present invention system.Part as the reverse primer complementary strand that will produce otch should be designed to the derivative in conjunction with dNTP, stops double-strand break to produce a kind of antagonism of nuclease by restriction enzyme.

Also rna polymerase promoter may be inserted reverse primer.Discern this promotor from transcribing of two ends by RNA polymerase among Fig. 6-(D) and carried out, closely similar with the model of previous application SDA in this case.

Synthetic nucleic acid of the present invention is strand, with regard to strand, is made of complementary nucleotide sequence, and its major part is base pairing.By utilizing this feature, can detect the synthetic product.By implementing the method for nucleic acid of the present invention, fluorochrome is being arranged as double-stranded specific intercalator (double-specific intercalater) ethidium bromide for example, SYBR Green I or Pico Green increase along with the increase of product can be observed intensity of fluorescence.By monitoring it, just may in closed system, follow the tracks of (real-time) building-up reactions in real time.Also can consider in PCR method to use the detection system of the type, but think that many problems are arranged, because can not distinguish the signal etc. of product signal and primer dipolymer.Yet, when the present invention uses this system, the ability that increases non-special base pairing is very low, therefore, estimate that highly sensitive and low the interference may can obtain simultaneously, double-stranded specific intercalator (double-specific intercalater) is similar to using, and can utilize the transfer of fluorescent energy to be used to realize the method for detection system in same system.

The method of nucleic acid of the present invention is supported by the complementary strand reaction of the synthetic strand displacement type of archaeal dna polymerase catalysis.Between the above-mentioned reaction period, also comprise the not reactions steps of essential strand displacement type polysaccharase.Yet, for simplification and the economic viewpoint of forming reagent, use a kind of archaeal dna polymerase favourable, this kind archaeal dna polymerase, following enzyme is known.In addition, can utilize the various mutant of these enzymes in the scope of the invention, they all have the active and strand displacement activity of the complementary strand of being used for synthetic sequence-dependence.Wherein mutant refers to and comprises that those only have the structure that causes the required catalytic activity of enzyme or those are by for example suddenling change to catalytic activity the mutant of the modification that stability or thermostability are carried out in the amino acid.

The Bst archaeal dna polymerase

Bca (exo-) archaeal dna polymerase

Dna polymerase i Ke Lienuo (Klenow) fragment

The Vent archaeal dna polymerase

Vent (Exo-) archaeal dna polymerase (the Vent archaeal dna polymerase that lacks exonuclease activity)

Deep Vent archaeal dna polymerase

Deep Vent (Exo-) archaeal dna polymerase (the Deep Vent archaeal dna polymerase that lacks exonuclease activity)

Φ 29 phage archaeal dna polymerases

MS-2 phage archaeal dna polymerase

Z-Taq archaeal dna polymerase (precious wine is made)

KOD archaeal dna polymerase (Japan twists flax fibers and weave)

In these enzymes, Bst archaeal dna polymerase and Bca (exo-) archaeal dna polymerase is required especially enzyme, because they have to a certain degree thermostability and high catalytic activity.In preferred embodiments, reaction of the present invention can isothermally realize, but because the adjusting (Tm) of melting temperature(Tm) etc. can not always can utilize temperature required condition to keep the stable of enzyme.Therefore, it is one of thermally-stabilised required condition of enzyme.Although isothermal reaction is feasible, thermally denature can provide nucleic acid as initial template, and in this respect, the selection of testing program has been widened in the use of thermophilic enzyme.

Vent (Exo-) archaeal dna polymerase is the enzyme that existing strand displacement activity has high heat stability again.The known complementary strand synthesis reaction that relates to by the archaeal dna polymerase strand displacement obtain promoting by adding single strand binding protein (Paul M.Lizardi et al., Nature Genetics, 19, 225-232, July, 1998).Use this reaction among the present invention, and, can estimate to promote the required effect of synthetic complementary strand by adding single strand binding protein.For example, T4 gene 32 is effective as Vent (Exo-) archaeal dna polymerase single strand binding protein.

Because archaeal dna polymerase does not have 3 '-5 ' exonuclease activity, the synthetic of complementary strand can not stop at template 5 '-end, causes producing the outstanding of a base, and this is known phenomenon.This phenomenon of the present invention is not preferred, because when the synthetic arrival of complementary strand is terminal, 3 '-end causes starting the synthetic of next complementary strand.Yet, add base " A " to 3 '-end by the high probability of archaeal dna polymerase, select sequence so that,, also can not have problems if add wrong extra base by dATP since 3 '-terminal synthesizing from " A ".In addition, during the complementary strand synthetic in addition 3 '-end be highlighted, utilize that the activity digestion of 3 ' → 5 ' exonuclease is outstanding to make it become blunt ends.For example, because the Vent archaeal dna polymerase has such activity, this enzyme can mix use with (Exo-) archaeal dna polymerase to solve described problem.

The present invention synthesizes or the necessary all ingredients of amplification of nucleic acid can be packed in advance, and provide with the form of test kit, particularly, test kit provided by the present invention comprises as synthetic complementary strand synthetic primer and the necessary various oligonucleotide of outer primer that are used for replacement(metathesis)reaction, be used for complementary strand synthetic substrate dNTP, be used to realize strand displacement type complementary strand synthetic archaeal dna polymerase, for enzyme reaction provides the damping fluid of conditions suitable and is used to detect the necessary medium of building-up reactions product.Particularly, in the preferred pattern of the present invention, need not the reagent that adds between the reaction period, and, wherein only just can start this reaction by the adding sample thus for moving into essential reagent of supplying with of reaction institute behind the reaction vessel.By utilize luminous signal or fluorescent signal can be in container the system of detection reaction product.Needn't open and close container after the reaction.This for pre-anti-pollution be very desirable.

The present invention is synthetic to have complementary nucleotide sequence and alternately is connected single intrachain nucleic acid.Purposes below this nucleic acid for example has: first feature is an advantage of utilizing the ad hoc structure that has complementary sequence in a part to bring, this feature is estimated to help detecting, the system that becomes known for detecting nucleic acid is promptly arranged, and wherein the signal of its variation depends on and the complementary nucleotide sequence base pairing.For example, by being used in combination the method for double-stranded specific intercalator as aforesaid detection reagent, the detection system that makes full use of synthetic product feature of the present invention can be achieved.If the product of building-up reactions of the present invention in described detection system a thermally denature takes place, and turns back to original temperature, intramolecularly annealing is preferential to be taken place, and therefore allows base pairing fast between the complementary sequence.If there is non-specific product, thus in the molecule they do not have complementary sequence make by thermally denature be separated into 2 or more molecule after, they can not just turn back to original two strands at once.By in the thermally denature step that detects the prerequisite confession, follow the interference of non-specific responding to be minimized.The terminated meaning if used not heat resistanceheat resistant of archaeal dna polymerase, thermally denature step respond, and therefore help control reaction temperature.

Second feature is the ring that usually forms the energy base pairing.The structure of the ring of energy base pairing shows in Fig. 4.Be made of the nucleotide sequence between nucleotide sequence F2c (X2c) and the insertion F2c-F1c (X1c) as this ring of seeing among Fig. 4, F2c (X2c) can carry out primer annealing.Sequence between F2c-F1c (or between more general form X2c-X1c) is the Nucleotide derived sequence that comes from template.Therefore, if having the probe and the hybridization of this district of complementary nucleotide sequence, the specific detection of template is feasible.In addition, but this usually base pairing of district, and therefore, thermally denature needn't be carried out prior to hybridization.The Nucleotide of forming the ring in the amplified reaction product of the present invention has length arbitrarily.Therefore, if wish and probe hybridization, the district that will be annealed by primer and design respectively to stop their competition by the district that probe will be hybridized wherein may obtain the ideal response condition.

The preferred pattern according to the present invention is supplied with the ring of energy base pairing in a large number in single-chain nucleic acid.This means a large amount of probes can with a part nucleic acid hybridization to allow highly sensitive detection.Therefore not only may realize improving sensitivity also may realize based on the special reaction principle for example congregation detect the method for nucleic acid.For example, be fixed on fine granular for example the probe on the polystyrene latex join in the reaction product of the present invention, the congregation of observing latex particle is product and probe hybridization.The intensity of congregation just can be carried out highly sensitive and quantitative observation by optical detecting.Perhaps also can observe congregation, so also can set up reactive system without the optical determinator by bore hole.

In addition, reaction product of the present invention is allowed many bonded marks of wanting, and wherein every nucleic acid molecule can carry out chromatography and detect.In the immunoassay field, the actual application is to utilize the visible certification mark to use the analytical procedure (immunochromatography) of chromatography media.This method is clipped in the antibody that is fixed on the chromatography media and the principle between traget antibody based on analyte, and unreacted marked member is by wash-out.Reaction product of the present invention is applied on the foranalysis of nucleic acids this principle.Just, preparation is at the label probe of loop section and be fixed on the chromatography media to catching the preparation capture probe, analyzes to allow in chromatography media.Sequence and loop section complementary capture probe are utilized, because reaction product of the present invention has a large amount of rings, product combines to provide the naked eyes discernible signal with the probe of a large amount of marks.

Reaction product of the present invention usually can be supplied with the ring district of base pairing, can widen other various detection systems.For example, the system that utilizes surperficial cytogene group to use stationary probe to detect this loop section is feasible.In addition, if, just can carry out more sensitive fluorometric analysis with the probe of double-stranded special this loop section of insert mark.Or the ability of actively utilizing nucleic acid of the present invention 3 '-and 5 '-side with form can base pairing ring.For example, design a ring and make it between normal type and undesired type, the common nucleotide sequence be arranged, make it produce difference therein and design other ring.Confirm that by probe there is gene in common ground, and when other district's confirmation has undesired the existence, might the composition characteristic analytical system.Because the reaction of nucleic acid of the present invention also can isothermally be carried out, the advantage of being worth mentioning is that can carrying out in real time by general fluorophotometer, (real-time) analyzes.Heretofore, it is known wanting the structure of annealed nucleic acid in the same chain.Yet, by the present invention obtain to have the nucleic acid that nucleotide sequence alternately is connected in the strand be new because it comprise a large amount of can with the ring of other base pairing.

On the other hand, a large amount of ring self of giving by reaction product of the present invention can be used as probe, for example, in the DNA chip, probe high-density in limited zone is piled up, and can be fixed on certain regional oligonucleotide limited amount in this technology, therefore by utilizing product of the present invention can be fixed by high-density by the annealed probe in a large number, be that reaction product of the present invention can be used as the fixed probe on the DNA chip, amplification afterreaction product can be fixed by any technology known in the art, or, cause generating the fixation reaction product with the oligonucleotide of fixed oligonucleotide as amplified reaction of the present invention.Therefore by using the fixed probe, a large amount of sample DNAs are hybridized in limited zone, and the result estimates to obtain high signal.

The accompanying drawing summary

Fig. 1 is the diagram of reaction principle (1)-(4) part in the preferred pattern of the present invention.

Fig. 2 is the diagram of reaction principle (5)-(7) part in the preferred pattern of the present invention.

Fig. 3 is the diagram of reaction principle (8)-(10) part in the preferred pattern of the present invention.

Fig. 4 is the diagram by the formed ring structure of single-chain nucleic acid of the present invention.

Fig. 5 is the diagram of (A)-(B) part in the basic pattern of the present invention.

Fig. 6 is the diagram of (C)-(D) part in the basic pattern of the present invention.

Fig. 7 is the position relation that shows each nucleotide sequence of forming oligonucleotide in the M13mp18 target nucleotide sequences.

Fig. 8 is the photo that shows by being the product agarose electrophoresis result that obtains of the method for the synthetic single-chain nucleic acid of the present invention of template with M13mp18.

Swimming lane 1:XIV molecular weight marker

Swimming lane 2:1fmol M13mp18 dsDNA

Swimming lane 3: do not have target

Fig. 9 is the photo that shows the agarose gel electrophoresis result of restriction enzyme digestion product, and wherein said product obtains in embodiment 1 by nucleic acid building-up reactions of the present invention.

Swimming lane 1:XIV molecular weight marker

Swimming lane 2: the BamHI digestion of purified product

Swimming lane 3: the PvuII digestion of purified product

Swimming lane 4: the HindIII digestion of purified product

Figure 10 is the photo that shows product agarose gel electrophoresis result, the method that described product is in the presence of trimethyl-glycine is template with M13mp18 by synthesizing single-stranded nucleic acid of the present invention obtains, 0,0.5,1 and 2 are meant the concentration (M) that adds the trimethyl-glycine in the reaction soln, the negative contrast of N ,-21 are meant that the concentration of template DNA is 10 ^-21Mol.

Figure 11 is the position relation that shows each nucleotide sequence of forming oligonucleotide from HVB derives next target nucleotide sequences.

Figure 12 is the photo of demonstration by the product agarose gel electrophoresis result of the method acquisition of synthesizing single-stranded nucleic acid of the present invention, and the HBV-M13mp18 that wherein is combined among the M13mp18 is a template.

Swimming lane 1:XIV molecular weight marker

Swimming lane 2:1fmol HBV-M13mp18 dsDNA

Swimming lane 3: do not have target

Figure 13 is the photo of demonstration by the alkaline denaturation product agarose gel electrophoresis result of the method acquisition of synthesizing single-stranded nucleic acid of the present invention,

Swimming lane 1: from the HindIII-digestion fragment of lambda phage

Reaction product among swimming lane 2: the embodiment 1

Reaction product among swimming lane 3: the embodiment 3

Figure 14 is the product agarose gel electrophoresis result's that shows that the method by synthesizing single-stranded nucleic acid of the present invention obtains a photo, and the concentration of its M13mp18 that hits changes, above show the result who reacted 1 and 3 hour respectively with following photo.

Swimming lane 1:M13mp18 dsDNA 1 * 10 ^-15The mol/ pipe

Swimming lane 2:M13mp18 dsDNA 1 * 10 ^-16The mol/ pipe

Swimming lane 3:M13mp18 dsDNA 1 * 10 ^-17The mol/ pipe

Swimming lane 4:M13mp18 dsDNA 1 * 10 ^-18The mol/ pipe

Swimming lane 5:M13mp18 dsDNA 1 * 10 ^-19The mol/ pipe

Swimming lane 6:M13mp18 dsDNA 1 * 10 ^-20The mol/ pipe

Swimming lane 7:M13mp18 dsDNA 1 * 10 ^-21The mol/ pipe

Swimming lane 8:M13mp18 dsDNA 1 * 10 ^-22The mol/ pipe

Swimming lane 9: do not have target

Swimming lane 10:XIV molecular weight marker

Figure 15 shows that the position that sudden change position and each are distinguished with respect to target nucleotide sequences (target) concerns that the guanine of underscore is replaced by VITAMIN B4 in the sudden change.

Figure 16 is the photo that shows amplified reaction product agarose gel electrophoresis result of the present invention.

M:100bp ladder (New England Biolabs)

N: do not have template (purified water)

WT:1fmol wild-type template M13mp18

MT:1fmol sudden change template M13mp18FM

Figure 17 is a position relation of forming each nucleotide sequence of oligonucleotide in the nucleotide sequence of code displaying said target mrna.

Figure 18 shows with mRNA to be the photo of template by the product agarose gel electrophoresis result of the method acquisition of synthesizing single-stranded nucleic acid of the present invention.

Implement the best pattern of the present invention

The amplification in the zone among 1 couple of M13mp18 of embodiment

The present invention is synthetic to have method that complementary strand alternately is connected to the nucleic acid in the strand to utilize M13mp18 is that template is attempted, using four kinds of primers in the experiment is M13FA, M13RA, M13F3, and M13R3, M13F3 and M13R3 are the outer primers of replacing respectively article one nucleic acid that obtains for synthetic starting point with M13FA and M13RA.Because with the synthetic back of M13FA (or M13RA) outer primer is the primer of the synthetic starting point of complementary strand.Close on packing phenomenon by utilization these are designed to be annealed to the district of closing on M13FA (or M13RA).In addition, these primers are set to high density the annealing of M13FA (or M13RA) are preferentially taken place.

The nucleotide sequence of forming each primer is shown in sequence table, and the constitutional features of primer is summarized below.In addition, close to tie up among Fig. 7 at the position in each district of target nucleotide sequences (target) and show.

The district of district/3 ' of primer 5 '-side-side

M13FA with by F1c district in the M13FA synthetic complementary strand identical/with M13mp18

Middle F2c district is complementary

M13RA with by R1c district in the M13RA synthetic complementary strand identical/with pass through M13FA

The R2c district is complementary in the synthetic complementary strand

The F3c complementation that F2c district 3 '-side is closed among M13F3 and the M13mp18

The R3c complementation that M13R3 closes on by F2c district 3 '-side in the M13FA synthetic complementary strand

By described primer, nucleic acid wherein extend to the district of R1c among the M13mp18 from F1c, and complementary nucleotide sequence is alternately to be connected in the strand by the one-tenth ring sequence that contains F2c.Shown in below being combined in of the reaction soln of the method by the synthetic nucleic acid of the present invention of these primers.

Reaction soln combination (among the 25 μ L)

20mM?Tris-HCl?pH8.8

10mM?KCl

10mM(NH ₄) ₂SO ₄

6mM?MgSO ₄

0.1％Triton?X-100

5% dimethyl sulfoxide (DMSO) (DMSO)

0.4mM?dNTP

Primer:

800nM?M13FA/SEQ?ID?NO：1

800nM?M13RA/SEQ?ID?NO：2

200nM?M13F3/SEQ?ID?NO：3

200nM?M13R3/SEQ?ID?NO：4

Target: M13mp18 dsDNA/SEQ ID NO:5

Reaction: above-mentioned reaction soln was 95 ℃ of heating 5 minutes, this target sex change becomes strand, reaction soln is transferred in the ice-cold water, add 4 U Bst archaeal dna polymerases (NEW ENGLANDBiolabs), mixture was in 65 ℃ of reactions 1 hour, after the reaction, stopped this reaction in 10 minutes in 80 ℃, forward to again in the ice-cold water then.

The confirmation of reaction: 1 μ l sample-loading buffer is added in the reaction soln above the 5 μ l, and sample was gone up electrophoresis 1 hour in 80mV at 2% sepharose (0.5%TBE).As molecular weight marker, (Molecular Probes, Inc.) gel behind the dyeing electrophoresis is with checking nucleic acid with SYBR Green I with XIV (100bp ladder, Boehringer Mannheim).The result shows that in Fig. 8 each swimming lane is respectively with respect to following sample.

1.XIV molecular weight marker

2.1?fmol?M13mp18?dsDNA

3. there is not target

In the swimming lane 3, except unreacted primer be colored be confirmed, band is not proved.There is target in the swimming lane 2, confirms that product is low-molecular-weight ladder-tape, be unsharp smear in the dyeing of high molecular place, and band is difficult in electrophoresis in the glue.In the lower molecular weight band, product desired near 290bp and the 450bp band and the building-up reactions of the present invention is consistent, promptly double-stranded SEQ ID NO:11 and 12 (two strands that is equivalent to formation shown in Fig. 2-(7) and the 2-(10)) and strand SEQ ID NO:13 (being equivalent to Fig. 3-(9) long strand).Therefore confirm reaction carrying out according to expectation.Because this reaction is that successive reaction is to allow the molecular weight of reacting condition product basically, further because this product has part strand and double-stranded compound complex construction, so expectation can cause producing at the high molecular place unintelligible smear pattern and not by the result of electrophoretic band.

Embodiment 2 confirms reaction product by the digestion of restriction enzyme

To have the nucleic acid construct that complementary nucleotide sequence alternately is connected the single intrachain embodiment of the invention 1 acquisition clearly in order explaining, to use the restriction enzyme digestion product.If by the fragment on the digestible energy generative theory, while does not exist (disappear) to produce unintelligible smear pattern as observed high molecular place among the embodiment 1 and not by electrophoretic band, just can estimate that any of these product alternately is connected single intrachain nucleic acid for the present invention has complementary sequence.

8 tube reaction solution among the embodiment 1 (200 μ l) are by being deposited and purifying with phenol processing and alcoholic acid precipitating action, reclaiming the precipitation that produces also is dissolved in the 200 μ l TE damping fluids again, use restriction enzyme BamHI respectively, PvuII, with HindIII in 37 ℃ of digestion 10 μ l aliquots containigs 2 hours, digest was gone up electrophoresis 1 hour in 80mV at 2% sepharose (0.5%TBE).(Gensura Laboratories, Inc.) as molecular weight marker, (Molecular Probes, Inc.) gel behind the dyeing electrophoresis is with checking nucleic acid with SYBR Green I with Super Ladder-Low (100bp ladder).The result shows that in Fig. 9 each swimming lane is respectively with respect to following sample.

The 1:XIV molecular weight marker

2: the BamHI digestion of purified product

3: the PvuII digestion of purified product

4: the HindIII digestion of purified product

The nucleotide sequence of estimating the relevant short amplified production of composition is those SEQ ID NO:13,14,15 and 16, from these nucleotide sequences, expectation is as shown in table 1 with each segmental size of restriction enzyme digestion, and " L " is meant because L is the fragment (strand) that contains ring and therefore do not determine the electrophoresis position in the table.

The postdigestive fragment of table 1. amplified production restriction enzyme of the present invention

(11,15; Do not confirm)

Because nearly all band is converted into required band before the digestion, this just confirms that the goal response product has obtained amplification, and showing does not in addition have or seldom have non-specific product.

Embodiment 3 adds the promoter action of trimethyl-glycine to amplified reaction

Detect trimethyl-glycine (N, N, N-Trimethyl glycine, Sigma) experiment that nucleic acid amplification reaction is influenced added in the reaction soln.Similar to embodiment 1, under the situation that has various concentration trimethyl-glycines, be that template is synthesized the present invention and had complementary strand and alternately be connected single intrachain nucleic acid with M13mp18.Identical among the used primer of this experiment and the embodiment 1, the amount of template DNA is 10 ^-21Mol (M13mp18), water is as negative control.Add concentration and be 0,0.5,1 and the 2M trimethyl-glycine in reaction soln, the combination of reaction soln is as follows.

The composition of reaction soln (among the 25 μ L)

20mM?Tris-HCl?pH8.8

4mM?MgSO ₄

0.4mM?dNTPs

10mM?KCl

10mM(NH ₄) ₂SO ₄

0.1％Triton?X-100

Primer:

800nM?M13FA/SEQ?ID?NO：1

800nM?M13RA/SEQ?ID?NO：2

200nM?M13F3/SEQ?ID?NO：3

200nM?M13R3/SEQ?ID?NO：4

Target: M13mp18 dsDNA/SEQ ID NO:5

Polysaccharase, reaction conditions, and reacted deposition condition is with identical described in the embodiment 1.

The result shows in Figure 10, and the concentration of trimethyl-glycine is 0.5 or 1.0M in the reaction, and the amount of amplified production obtains increasing.And, if its concentration is increased to 2.0M, then do not observe amplification.The trimethyl-glycine of suitable concn thus promotes amplified reaction.The reason that the amplified production amount reduces is estimated as when the concentration of trimethyl-glycine is 2.0M, and it is too many that Tm is fallen.

The amplification of embodiment 4 HBV gene orders

Attempt the method for synthetic nucleic acid of the present invention, the partial sequence and its bonded M13mp18 that wherein have the HBV gene are used as template.Used 4 kinds of primer HB65FA (SEQ ID NO:6) in the experiment, HB65RA (SEQ ID NO:7), HBF3 (SEQ ID NO:8) and HBR3 (SEQ ID NO:9).HBF3 and HBR3 are the outer primers of replacing respectively article one nucleic acid that obtains for synthetic starting point with HB65FA and HB65RA.Because with the synthetic back of HB65FA (or HB65RA) outer primer is the primer of the synthetic starting point of complementary strand.Close on packing phenomenon by utilization these are designed to be annealed to the district of closing on HB65FA (or HB65RA).In addition, these primers are set to high density the annealing of HB65FA (or HB65RA) are preferentially taken place.HBV's target sequence among this embodiment (430bp) derives among the M13mp18 by being combined in, and shows in SEQ ID NO:10.

The nucleotide sequence of forming each primer is shown in sequence table, and the constitutional features of each primer is summarized below.In addition, close to tie up among Figure 11 at the position in each district of target nucleotide sequences (target) and show

The district of district/3 ' of primer 5 '-side-side

HB65FA with by F1c district in the HB65FA synthetic complementary strand identical/with

The F2c district is complementary among the HBV-M13mp18

HB65RA with by R1c district in the HB65RA synthetic complementary strand identical/with pass through

The R2c district is complementary in the HB65FA synthetic complementary strand

The F3c complementation that F2c district 3 '-side is closed among HBF3 and the HBV-M13mp18

The R3c that HBR3 closes on by F2c district 3 '-side in the HB65FA synthetic complementary strand is mutual

Mend

By described primer, nucleic acid, wherein have section H BV gene and its bonded M13mp18HBV-M13mp18) in extend to the district of R1c from F1c, and complementary nucleotide sequence is that the one-tenth ring sequence that alternately contains F2c by insertion alternately is connected in the strand.Except above-mentioned the primer, this be reflected at embodiment 1 in carried out under the identical condition.Reaction soln is by the agarose gel electrophoresis analysis, and the result shows in Figure 12.Each swimming lane is respectively with respect to following sample.

1.XIV molecular weight marker

2.1fmol?HBV-M13mp18?dsDNA.

3. there is not target

Similar to embodiment 1, under the situation that target exists, only confirm that product is low-molecular-weight ladder-tape, in high molecular place dyeing for unsharp smear be difficult in the glue in electrophoretic band (in the swimming lane 2) the lower molecular weight band, product desired near 310bp and the 480bp band and the building-up reactions of the present invention is consistent, promptly double-stranded SEQ ID NO:17 and 18.Therefore confirm reaction carrying out according to expectation.Result as described in example 1 above, the feature structure of estimating synthetic product of the present invention cause producing at the high molecular place unintelligible smear pattern and not by electrophoretic band.From this experiment, confirm even increasing that the present invention can utilize with different sequences (target).

Embodiment 5 confirms the size of building-up reactions product

In order to confirm that synthetic nucleic acid construct of the present invention is by its length of electrophoretic analysis under the alkaline denaturation condition, in the presence of hitting, embodiment 1 or 4 in 5 each reaction soln of μ l, adds 1 μ l sample-loading buffer, sample was gone up electrophoresis 14 hours in 50mA at 0.7% sepharose (50mM NaOH, 1mM EDTA).With the lambda phage fragment of HindIII-digestion as molecular weight marker, the 1M Tris of the gel behind the electrophoresis, the pH8 neutralization and with SYBR Green I (Molecular Probes Inc.) dyes to verify nucleic acid.The result shows that in Figure 13 each swimming lane is respectively with respect to following sample.

1: from the HindIII-digestion fragment of lambda phage

2: the reaction product among the embodiment 1

3: the reaction product among the embodiment 4

Electrophoresis reaction product under the alkaline denaturation condition can confirm the size in the strand state.The size that confirms embodiment 1 (swimming lane 2) and embodiment 4 (swimming lane 3) primary product within 2kbase, and, demonstrate product of the present invention by this analysis and in the scope that can analyze confirmation, be extended the size that is not less than 6kbase at least.In addition, under denatured state, be not separated into single littler strand among embodiment 1 and the embodiment 4 under the not sex change condition by electrophoretic band.

Depend on the confirmation of the amplification of target level in the reaction in the zone among 6 pairs of amplifications of embodiment M-13mp13

Can observe the influence of the involutory one-tenth of the target nucleic acid of the present invention of change in concentration.Except the amount as the M13mp18 dsDNA of target is 0-1 fmol and the reaction times is that the method for nucleic acid of the present invention under the identical condition of 1 or 3 hour external and embodiment 1 is implemented.Similar to embodiment 1, sample is gone up electrophoresis at 2% sepharose (0.5%TBE), and (Molecular Probes, Inc.) dyeing is with checking nucleic acid with SYBR Green I.With XIV (100bp ladder, Boehringer Mannheim) as molecular weight marker, the result in Figure 14 (above: reacted 1 hour, below: reacted 3 hours) show that each swimming lane is respectively with respect to following sample:

1.M13mp18 dsDNA 1 * 10 ^-15The mol/ pipe.

2.M13mp18 dsDNA 1 * 10 ^-16The mol/ pipe.

3.M13mp18 dsDNA 1 * 10 ^-17The mol/ pipe.

4.M13mp18 dsDNA 1 * 10 ^-18The mol/ pipe.

5.M13mp18 dsDNA 1 * 10 ^-19The mol/ pipe.

6.M13mp18 dsDNA 1 * 10 ^-20The mol/ pipe.

7.M13mp18 dsDNA 1 * 10 ^-21The mol/ pipe.

8.M13mp18 dsDNA 1 * 10 ^-22The mol/ pipe.

9. there is not target.

10.XIV molecular weight marker.

Be with jointly in each swimming lane in electrophorogram to show, be the unreacted primer that dyed look than lower part.Do not consider the reaction times, when lacking target, then can not observe amplified production.The dyeing pattern depends on the concentration of target, only could obtain the dyeing pattern of amplified production in the presence of target.Can confirm that the concentration of amplified production is lower when in addition, increasing the reaction times.

The detection of embodiment 7 point mutation

(1) preparation of M13mp18FM (mutant)

Used target DNA is M13mp18 (wild-type) and M13mp18FM (mutant), in order to prepare mutant M13mp18FM, uses LA PCR ^TMVitro mutagenesis test kit (the wine treasured is made) is replaced a Nucleotide and is obtained sudden change.After this, confirm this sequence by order-checking.The sequence in F1 district is as follows:

Wild-type: CCGGGGATCCTCTAGAGTCG (SEQ ID NO:19)

Mutant: CCGGGGATCCTCTAGAGTCA (SEQ ID NO:20)

(2) design of primers

Wild-type provides different nucleotide sequences at F1c district 5 '-end respectively with the used FA primer of mutant, the position of sudden change and close to tie up among Figure 15 at every zone position of target nucleotide sequences (target) and show.

(3) amplified reaction

As follows by utilizing M13mp18 (wild-type) and M13mp18FM (mutant) to carry out the experiment whether reaction of detection specificity template amplification takes place for template applications binding specificity primer.

Wild-type amplification the primer: FAd4, RAd4, F3, R3

Mutant amplification the primer: FAMd4, RAd4, F3, R3

Each primer nucleotide sequence is as follows:

FAd4：CGACTCTAGAGGATCCCCGGTTTTTGTTGTGTGGAATTGTGAGCGGAT(SEQ?ID?NO：21)

FAMd4：TGACTCTAGAGGATCCCCGGTTTTTGTTGTGTGGAATTGTGAGCGGAT(SEQ?ID?NO：22)

RAd4：CGTCGTGACTGGGAAAACCCTTTTTGTGCGGGCCTCTTCGCTATTAC(SEQ?ID?NO：23)

F3：ACTTTATGCTTCCGGCTCGTA(SEQ?ID?NO：24)

R3：GTTGGGAAGGGCGATCG(SEQ?ID?NO：25)

(4) detect point mutation among the M13mp18

The reaction soln combination is as follows:

Final concentration

D2W 3.75μL

10X?Thermo?pol?buffer(NEB) 2.5μL 20mM?Tris-HCl?pH8.8

10mM?KCl

10mM(NH ₄) ₂SO ₄

6mM?MgSO ₄

0.1％TritonX-100

2.5mM?dNTP 4μL 400μM

100mM?MgSO ₄ 0.5μL

4?M?Betaine 6.25μL 1M

M13FAd4 primer (10pmol/ μ L) or

M13FAMd4 primer (10pmol/ μ L) 2 μ L 800nM

M13RAd4 primer (10pmol/ μ L) 2 μ L 800nM

M13F3 primer (10pmol/ μ L) 0.5 μ L 200nM

M13R3 primer (10pmol/ μ L) 0.5 μ L 200nM

Total amount 22 μ L

1fmol (2 μ l) target M13mp18 or M13mp18FM are joined in the reaction soln in 95 ℃ of heating 5 minutes, and wherein said target sex change becomes strand.Reaction soln is transferred in the ice-cold water, adding 1 μ l (8U) Bst archaeal dna polymerase (NEW ENGLAND BioLabs) reacted 1 hour in 68 ℃ or 68.5 ℃, after the reaction, stopped this reaction in 10 minutes in 80 ℃, reaction soln forwards in the ice-cold water again.

As shown in figure 16, when being used as the FA primer, only under wild pattern plate situation, observe effective amplification for wild-type FAd4.On the other hand, when being used as the FA primer, only under mutant template situation, observe effective amplification for mutant FAMd4.

Show by utilizing amplified reaction of the present invention check point sudden change effectively from The above results.

Embodiment 8 is the amplified reaction of target with mRNA

By utilizing with mRNA is that the method for the synthetic nucleic acid of the present invention of target nucleic acid is attempted.Be the preparation said target mrna, the prostate cancer cell line LNCaP cell (ATCC No.CRL-1740) that will express prostate specific antigen (PSA) is that K562 cell (ATCC No.CCL-243) was at 1: 10 with the chronic bone marrow leukemia cells of non-expressing cell ⁶By 100: 10 ⁶Scope is mixed, and then by being used to the test kit from the RNeasy Mini of Qiagen (Germany), extracts total RNA, and used 4 kinds of primers are PSAFA in the experiment, PSARA, PSAF3 and PSAR3.PSAF3 and PSAR3 are the outer primers of replacing respectively first nucleic acid that obtains for synthetic starting point with PSAFA and PSARA.In addition, the annealing of PSAFA (or PSARA) preferentially takes place the concentration of these primers to be provided with height.The nucleotide sequence of forming each primer is as follows,

Primer:

PSAFA：TGTTCCTGATGCAGTGGGCAGCTTTAGTCTGCGGCGGTGTTCTG(SEQ?ID?NO：26)

PSARA：TGCTGGGTCGGCACAGCCTGAAGCTGACCTGAAATACCTGGCCTG(SEQ?ID?NO：27)

PSAF3：TGCTTGTGGCCTCTCGTG(SEQ?ID?NO：28)

PSAR3：GGGTGTGGGAAGCTGTG(SEQ?ID?NO：29)

The constitutional features of primer is summarized below.In addition, show in Figure 17 at the position relation and the restriction enzyme Sau3AI recognition site of coding for each primer of dna nucleotide sequence of said target mrna.

The district of district/3 ' of primer 5 '-side-side

PSAFA with by F1c district in the PSAFA synthetic complementary strand identical/with the target nucleotide preface

The F2c district is complementary in the row

PSARA with by R1c district in the PSARA synthetic complementary strand identical/with pass through PSAFA

The R2c district is complementary in the synthetic complementary strand

The F3c complementation that F2c district 3 '-side is closed in PSAF3 and the target nucleotide sequences

The R3c complementation that PSAR3 closes on by F2c district 3 '-side in the PSAFA synthetic complementary strand

The combination of the reaction soln of nucleic acid method of the present invention is as follows:

The composition of reaction soln (among the 25 μ L)

20mM?Tris-HCl?pH8.8

4mM?MgSO ₄

0.4mM?dNTPs

10mM?KCl

10mM(NH ₄) ₂SO ₄

0.1％Triton?X-100

0.8M?betaine

5mM?DTT

1600nM PSAFA﹠amp; The PSARA primer

200nM PSAF3﹠amp; The PSAR3 primer

8 U Bst archaeal dna polymerases

100?U?Rever?Tra?Ace(Toyobo?Co.，Ltd.，Japan)

The total RNA of 5 μ g

Used composition mixes on ice, be target sequence with mRNA (strand) in this experiment, so needn't obtain the step of strand by thermally denature.Reacted 45 minutes in 65 ℃, stopped this reaction in 5 minutes in 85 ℃, after the reaction, 5 μ l reaction solns electrophoresis on 2% sepharose is with SYBR Green I dyeing.

The result shows that in Figure 18 each swimming lane is respectively with respect to following sample:

Swimming lane Bst RT LNCaP cell count/10 ⁶The K562 cell count

1 - + 0

2 - + 10

3 + - 0

4 + - 10

5 + + 0

6 + + 1

7 + + 10

Digest 1 μ L equal portions reaction soln for Sau3AI in 8 swimming lanes 6

Digest 1 μ L equal portions reaction soln for Sau3AI in 9 swimming lanes 7

10 molecular weight markers, 100 bp ladder (New England Biolabs)

When lacking Bst archaeal dna polymerase or Rever Tra Ace, can not obtain amplified production (swimming lane 1-4).Described two kinds of enzymes all exist under the situation, if exist from the LNCaP derived RNA, then can detect amplified production (swimming lane 5-7).Can detect RNA (swimming lane 6) from LNCaP cell/1,000,000 a K562 cell extraction.When being positioned at the Sau3AI restriction enzyme sites digest amplification product of target inside, this product is digested the fragment (swimming lane 8 and 9) of estimated size.

From The above results, confirm in the method for nucleic acid of the present invention even can obtain required reaction product during for target with RNA.

Commercial Application

New oligonucleotides and the method for utilizing described oligonucleotides nucleic acid provide and have closed according to the present invention Become to have the method that complementary nucleotide sequence alternately is connected the nucleic acid in the strand, and without any need for multiple Assorted temperature control. Based on the present invention's complementary strand synthetic take oligonucleotides as primer with described template strand 3 '-Terminal starting point as synthetic new complementary strand is attended by the synthetic of ring that causes new primer annealing and reaches to close earlier The chain that becomes is the synthetic complementary strand product of template, is again passed through from the synthetic complementary strand institute of ring beginning But replace and base pairing. Obtain thus with from synthesizing as the synthetic nucleic acid of template and known nucleic acid For example SDA combination of method is of value to the combined coefficient of improving nucleic acid.

The further preference pattern according to the present invention provides the new method of nucleic acid, and the method can be improved The efficient of known method nucleic acid does not need complicated temperature control, and estimates to reach high efficiency Amplification, and can obtain high specific, be about to based on oligonucleotides of the present invention mutual as template strand and its Mend chain, wherein having the nucleic acid that complementary nucleotide sequence alternately is connected in the strand can be closed continuously Become. This reaction can proceed to exhausting of synthetic necessary initial product in theory. Continue synthetic from During the ring novel nucleic acids that begins to synthesize, carry out being replaced into of chain from the extension of the oligonucleotides that can be annealed into ring The extension of long single-chain nucleic acid (namely having many nucleic acid that complementary strand are connected this) provides 3 '-OH. The opposing party Face, 3 '-OH of long single-chain nucleic acid obtains thus with from the reaction of synthesizing complementary strand as template Extend, the synthetic new complementary strand that begins from ring is replaced, and this amplified reaction step is advanced under isothermy Row is also kept high specificity.

It is anti-to make oligonucleotides of the present invention can be used as nucleic acid when two close on the district according to designed design The primer of answering, this obviously helps specific conservative, by starting non-specific with non-specific mistake annealing The property the comparing with e.g.PCR of amplified reaction, no matter how the position relationship of required 2 primers just can hold Change places high specific required for the present invention is made explanations, can utilize this specificity high sensitivity and accurately inspection Survey SNPs.

The invention is characterized in by forming simple reagent just can easily obtain this reaction, for example originally The invention oligonucleotides has ad hoc structure, but this is the material of nucleotides sequence column selection, is simple few nuclear The thuja acid material. In addition, in preferred model, only by catalyzing and synthesizing substitutional complementary strand reaction

Dna polymerase reaction can be carried out. And, if the present invention take RNA as template, profit With archaeal dna polymerase Bca archaeal dna polymerase for example, not only has reverse transcriptase activity but also the strand displacement type is arranged Dna polymerase activity can be carried out by single enzyme all enzyme reactions, realizes height by simple enzyme The reaction principle of amplification of nucleic acid it be unclear that. Even synthetic anti-for the present invention being applied to known nucleic acid For example the combination for them needn't need extra enzyme among the SDA. And should with based on widow of the present invention The simple combination of nucleotides can be used in the various reaction systems, so the method for nucleic acid of the present invention exists The expense aspect also is favourable.

As mentioned above, the method for nucleic acid of the present invention and oligonucleotides wherein provide a kind of new former Reason has solved simultaneously many problems and has for example operated (needn't need temperature control), improves combined coefficient, economy Change and high specific.

Sequence table

＜110〉Eiken Chemical (Eiken Kagaku Kabushiki Kaisha)

＜120〉method of nucleic acid

<130>E2-001PCT2

<140>

<141>

<150>PCT/JP99/06213

<151>1999-11-08

<160>29

<170>PatentIn?Ver.2.0

<210>1

<211>52

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the primer sequence of synthetic

<400>1

cgactctaga?ggatccccgg?gtactttttg?ttgtgtggaa?ttgtgagcgg?at 52

<210>2

<211>51

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the primer sequence of synthetic

<400>2

acaacgtcgt?gactgggaaa?accctttttg?tgcgggcctc?ttcgctatta?c 51

<210>3

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the primer sequence of synthetic

<400>3

actttatgct?tccggctcgt?a 21

<210>4

<211>17

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the primer sequence of synthetic

<400>4

gttgggaagg?gcgatcg 17

<210>5

<211>600

<212>DNA

＜213〉phage M13mp18

<400>5

gcgcccaata?cgcaaaccgc?ctctccccgc?gcgttggccg?attcattaat?gcagctggca?60

cgacaggttt?cccgactgga?aagcgggcag?tgagcgcaac?gcaattaatg?tgagttagct?120

cactcattag?gcaccccagg?ctttacactt?tatgcttccg?gctcgtatgt?tgtgtggaat?180

tgtgagcgga?taacaatttc?acacaggaaa?cagctatgac?catgattacg?aattcgagct?240

cggtacccgg?ggatcctcta?gagtcgacct?gcaggcatgc?aagcttggca?ctggccgtcg?300

ttttacaacg?tcgtgactgg?gaaaaccctg?gcgttaccca?acttaatcgc?cttgcagcac?360

atcccccttt?cgccagctgg?cgtaatagcg?aagaggcccg?caccgatcgc?ccttcccaac?420

agttgcgcag?cctgaatggc?gaatggcgct?ttgcctggtt?tccggcacca?gaagcggtgc?480

cggaaagctg?gctggagtgc?gatcttcctg?aggccgatac?ggtcgtcgtc?ccctcaaact?540

ggcagatgca?cggttacgat?gcgcccatct?acaccaacgt?aacctatccc?attacggtca?600

<210>6

<211>63

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the primer sequence of synthetic

<400>6

ctcttccaaa?agtaaggcag?gaaatgtgaa?accagatcgt?aatttggaag?acccagcatc?60

cag 63

<210>7

<211>43

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the primer sequence of synthetic

<400>7

gtggattcgc?actcctcccg?ctgatcggga?cctgcctcgt?cgt 43

<210>8

<211>16

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the primer sequence of synthetic

<400>8

gccacctggg?tgggaa 16

<210>9

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the primer sequence of synthetic

<400>9

ggcgagggag?ttcttcttct?ag 22

<210>10

<211>430

<212>DNA

＜213〉hepatitis B virus

<400>10

ctccttgaca?ccgcctctgc?tctgtatcgg?gaggccttag?agtctccgga?acattgttca?60

cctcaccata?cagcactcag?gcaagctatt?ctgtgttggg?gtgagttaat?gaatctggcc?120

acctgggtgg?gaagtaattt?ggaagaccca?gcatccaggg?aattagtagt?cagctatgtc?180

aatgttaata?tgggcctaaa?aatcagacaa?ctattgtggt?ttcacatttc?ctgccttact?240

tttggaagag?aaactgtttt?ggagtatttg?gtatcttttg?gagtgtggat?tcgcactcct?300

cccgcttaca?gaccaccaaa?tgcccctatc?ttatcaacac?ttccggaaac?tactgttgtt?360

agacgacgag?gcaggtcccc?tagaagaaga?actccctcgc?ctcgcagacg?aaggtctcaa?420

tcgccgcgtc 430

<210>11

<211>293

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the sequence of synthetic

<400>11

acaacgtcgt?gactgggaaa?accctttttg?tgcgggcctc?ttcgctatta?cgccagctgg?60

cgaaaggggg?atgtgctgca?aggcgattaa?gttgggtaac?gccagggttt?tcccagtcac?120

gacgttgtaa?aacgacggcc?agtgccaagc?ttgcatgcct?gcaggtcgac?tctagaggat?180

ccccgggtac?cgagctcgaa?ttcgtaatca?tggtcatagc?tgtttcctgt?gtgaaattgt?240

tatccgctca?caattccaca?caacaaaaag?tacccgggga?tcctctagag?tcg 293

<210>12

<211>293

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the sequence of synthetic

<400>12

cgactctaga?ggatccccgg?gtactttttg?ttgtgtggaa?ttgtgagcgg?ataacaattt?60

cacacaggaa?acagctatga?ccatgattac?gaattcgagc?tcggtacccg?gggatcctct?120

agagtcgacc?tgcaggcatg?caagcttggc?actggccgtc?gttttacaac?gtcgtgactg?180

ggaaaaccct?ggcgttaccc?aacttaatcg?ccttgcagca?catccccctt?tcgccagctg?240

gcgtaatagc?gaagaggccc?gcacaaaaag?ggttttccca?gtcacgacgt?tgt 293

<210>13

<211>459

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the sequence of synthetic

<400>13

acaacgtcgt?gactgggaaa?accctttttg?tgcgggcctc?ttcgctatta?cgccagctgg?60

cgaaaggggg?atgtgctgca?aggcgattaa?gttgggtaac?gccagggttt?tcccagtcac?120

gacgttgtaa?aacgacggcc?agtgccaagc?ttgcatgcct?gcaggtcgac?tctagaggat?180

ccccgggtac?cgagctcgaa?ttcgtaatca?tggtcatagc?tgtttcctgt?gtgaaattgt?240

tatccgctca?caattccaca?caacaaaaag?tacccgggga?tcctctagag?tcgacctgca?300

ggcatgcaag?cttggcactg?gccgtcgttt?tacaacgtcg?tgactgggaa?aaccctggcg?360

ttacccaact?taatcgcctt?gcagcacatc?cccctttcgc?cagctggcgt?aatagcgaag?420

aggcccgcac?aaaaagggtt?ttcccagtca?cgacgttgt 459

<210>14

<211>458

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the sequence of synthetic

<400>14

cgactctaga?ggatccccgg?gtactttttg?ttgtgtggaa?ttgtgagcgg?ataacaattt?60

cacacaggaa?acagctatga?ccatgattac?gaattcgagc?tcggtacccg?gggatcctct?120

agagtcgacc?tgcaggcatg?caagcttggc?actggccgtc?gttttacaac?gtcgtgactg?180

ggaaaaccct?ggcgttaccc?aacttaatcg?ccttgcagca?catccccctt?tcgccagctg?240

gcgtaatagc?gaagaggccc?gcacaaaaag?ggttttccca?gtcacgacgt?tgtaaaacga?300

cggccagtgc?caagcttgca?tgcctgcagg?tcgactctag?aggatccccg?ggtaccgagc?360

tcgaattcgt?aatcatggtc?atagctgttt?cctgtgtgaa?attgttatcc?gctcacaatt?420

ccacacaaca?aaaagtaccc?ggggatcctc?tagagtcg 458

<210>15

<211>790

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the sequence of synthetic

<400>15

acaacgtcgt?gactgggaaa?accctttttg?tgcgggcctc?ttcgctatta?cgccagctgg?60

cgaaaggggg?atgtgctgca?aggcgattaa?gttgggtaac?gccagggttt?tcccagtcac?120

gacgttgtaa?aacgacggcc?agtgccaagc?ttgcatgcct?gcaggtcgac?tctagaggat?180

ccccgggtac?cgagctcgaa?ttcgtaatca?tggtcatagc?tgtttcctgt?gtgaaattgt?240

tatccgctca?caattccaca?caacaaaaag?tacccgggga?tcctctagag?tcgacctgca?300

ggcatgcaag?cttggcactg?gccgtcgttt?tacaacgtcg?tgactgggaa?aaccctggcg?360

ttacccaact?taatcgcctt?gcagcacatc?cccctttcgc?cagctggcgt?aatagcgaag?420

aggcccgcac?aaaaagggtt?ttcccagtca?cgacgttgta?aaacgacggc?cagtgccaag?480

cttgcatgcc?tgcaggtcga?ctctagagga?tccccgggta?ctttttgttg?tgtggaattg?540

tgagcggata?acaatttcac?acaggaaaca?gctatgacca?tgattacgaa?ttcgagctcg?600

gtacccgggg?atcctctaga?gtcgacctgc?aggcatgcaa?gcttggcact?ggccgtcgtt?660

ttacaacgtc?gtgactggga?aaaccctggc?gttacccaac?ttaatcgcct?tgcagcacat?720

ccccctttcg?ccagctggcg?taatagcgaa?gaggcccgca?caaaaagggt?tttcccagtc?780

acgacgttgt 790

<210>16

<211>789

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the sequence of synthetic

<400>16

cgactctaga?ggatccccgg?gtactttttg?ttgtgtggaa?ttgtgagcgg?ataacaattt?60

cacacaggaa?acagctatga?ccatgattac?gaattcgagc?tcggtacccg?gggatcctct?120

agagtcgacc?tgcaggcatg?caagcttggc?actggccgtc?gttttacaac?gtcgtgactg?180

ggaaaaccct?ggcgttaccc?aacttaatcg?ccttgcagca?catccccctt?tcgccagctg?240

gcgtaatagc?gaagaggccc?gcacaaaaag?ggttttccca?gtcacgacgt?tgtaaaacga?300

cggccagtgc?caagcttgca?tgcctgcagg?tcgactctag?aggatccccg?ggtaccgagc?360

tcgaattcgt?aatcatggtc?atagctgttt?cctgtgtgaa?attgttatcc?gctcacaatt?420

ccacacaaca?aaaagtaccc?ggggatcctc?tagagtcgac?ctgcaggcat?gcaagcttgg?480

cactggccgt?cgttttacaa?cgtcgtgact?gggaaaaccc?tttttgtgcg?ggcctcttcg?540

ctattacgcc?agctggcgaa?agggggatgt?gctgcaaggc?gattaagttg?ggtaacgcca?600

gggttttccc?agtcacgacg?ttgtaaaacg?acggccagtg?ccaagcttgc?atgcctgcag?660

gtcgactcta?gaggatcccc?gggtaccgag?ctcgaattcg?taatcatggt?catagctgtt?720

tcctgtgtga?aattgttatc?cgctcacaat?tccacacaac?aaaaagtacc?cggggatcct?780

ctagagt?cg 789

<210>17

<211>310

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the sequence of synthetic

<400>17

gtggattcgc?actcctcccg?ctgatcggga?cctgcctcgt?cgtctaacaa?cagtagtttc?60

cggaagtgtt?gataagatag?gggcatttgg?tggtctgtaa?gcgggaggag?tgcgaatcca?120

cactccaaaa?gataccaaat?actccaaaac?agtttctctt?ccaaaagtaa?ggcaggaaat?180

gtgaaaccac?aatagttgtc?tgatttttag?gcccatatta?acattgacat?agctgactac?240

taattccctg?gatgctgggt?cttccaaatt?acgatctggt?ttcacatttc?ctgccttact?300

tttggaagag 310

<210>18

<211>465

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the sequence of synthetic

<400>18

gtggattcgc?actcctcccg?ctgatcggga?cctgcctcgt?cgtctaacaa?cagtagtttc?60

cggaagtgtt?gataagatag?gggcatttgg?tggtctgtaa?gcgggaggag?tgcgaatcca?120

cactccaaaa?gataccaaat?actccaaaac?agtttctctt?ccaaaagtaa?ggcaggaaat?180

gtgaaaccac?aatagttgtc?tgatttttag?gcccatatta?acattgacat?agctgactac?240

taattccctg?gatgctgggt?cttccaaatt?acgatctggt?ttcacatttc?ctgccttact?300

tttggaagag?aaactgtttt?ggagtatttg?gtatcttttg?gagtgtggat?tcgcactcct?360

cccgcttaca?gaccaccaaa?tgcccctatc?ttatcaacac?ttccggaaac?tactgttgtt?420

agacgacgag?gcaggtcccg?atcagcggga?ggagtgcgaa?tccac 465

<210>19

<211>20

<212>DNA

<213>M13mp18

<400>19

ccggggatcc?tctagagtcg 20

<210>20

<211>20

<212>DNA

＜213〉M13mp18 mutant

<400>20

ccggggatcc?tctagagtca 20

<210>21

<211>48

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the primer sequence of synthetic

<400>21

cgactctaga?ggatccccgg?tttttgttgt?gtggaattgt?gagcggat 48

<210>22

<211>48

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the primer sequence of synthetic

<400>22

tgactctaga?ggatccccgg?tttttgttgt?gtggaattgt?gagcggat 48

<210>23

<211>47

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the primer sequence of synthetic

<400>23

cgtcgtgact?gggaaaaccc?tttttgtgcg?ggcctcttcg?ctattac 47

<210>24

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the primer sequence of synthetic

<400>24

actttatgct?tccggctcgt?a 21

<210>25

<211>17

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the primer sequence of synthetic

<400>25

gttgggaagg?gcgatcg 17

<210>26

<211>44

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the primer sequence of synthetic

<400>26

tgttcctgat?gcagtgggca?gctttagtct?gcggcggtgt?tctg 44

<210>27

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the primer sequence of synthetic

<400>27

tgctgggtcg?gcacagcctg?aagctgacct?gaaatacctg?gcctg 45

<210>28

<211>18

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the primer sequence of synthetic

<400>28

tgcttgtggc?ctctcgtg 18

<210>29

<211>17

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the primer sequence of synthetic

<400>29

gggtgtggga?agctgtg 17

Claims (11)

1. the method for nucleic acid, described nucleic acid have the complementary nucleotide sequence that alternately connects on one bar chain, described method comprises:

A) provide the step of nucleic acid, its 3 ' end have can with the F1c annealed F1 district on same the chain, and described nucleic acid can form ring after described F1 district and F1c annealing, described ring comprises the F2c district that can carry out base pairing, and wherein said nucleic acid is second nucleic acid that provides by following step;

I) annealing steps, make the annealing of first oligonucleotide and template F2c district, wherein 3 ' of this template end comprises the F2c district and is positioned at the F1c district of F2c district 5 ' side, 5 ' end of this template comprises the R1 district and is positioned at the R2 district of R1 district 5 ' side, wherein this first oligonucleotide comprises at least two district F2 and F1c, F1c links to each other with the 5 ' side of F2

F2: have with the district of F2c district complementary nucleotide sequence and

F1c: with the F1c district of F2c district 5 ' side on the template district of basic identical nucleotide sequence is arranged,

The step of ii) synthetic first nucleic acid, described first nucleic acid has and described template complementary nucleotide sequence, this step with the F2 of first oligonucleotide as the synthetic starting point,

Iii) utilize polysaccharase catalysis strand replacement reaction to replace ii) synthetic first nucleic acid in the step, wherein with template in F2c 3 ' side annealed, first outer primer as the synthetic starting point and

Iv) annealing steps makes the ii) R2c district annealing of the nucleic acid of winning of second oligonucleotide and step I, two district R2 and R1c below wherein this second oligonucleotide comprises at least, and R1c links to each other with the 5 ' side of R2,

R2:3 ' end region, its have with R2c district complementary nucleotide sequence and

R1c: with the R1c district of the first nucleic acid R2c district, 5 ' side the district of basic identical nucleotide sequence is arranged,

Then with described second oligonucleotide as the synthetic starting point, synthetic second nucleic acid, and utilize polysaccharase catalysis strand replacement reaction to replace this second nucleic acid, wherein with template in 3 ' side annealed, second outer primer of R2c as the synthetic starting point;

B) synthesis step is synthetic himself the complementary strand of template with second nucleic acid of step a), will be synthetic starting point with 3 of F1c annealed F1 ' is terminal;

C) annealing steps, make the F2c district annealing in the ring that forms by second nucleic acid in first oligonucleotide and the step a), then carry out synthesis step, with this first oligonucleotide is synthetic starting point, carry out complementary strand by polysaccharase catalysis strand displacement type complementary strand synthesis reaction and synthesize, with displacement step b) middle institute synthetic complementary strand; With

D) annealing steps by in the metathetical complementary strand, makes the R1 annealing of R1c district and same chain 3 ' end in step c),

Then carry out synthesis step, with described 3 ' end is synthetic starting point, carrying out complementary strand by polysaccharase catalysis strand displacement type complementary strand synthesis reaction synthesizes, with displacement step c) middle institute synthetic complementary strand, wherein synthetic starting point be same chain 3 ' end can with R1c district annealed R1 district, and,, form the ring that contains the R2c district by the annealing of R1c and R1c.

2. the method for claim 1, the relation of melting temperature(Tm) below existing under the identical rigorous condition in the described reaction between used every kind of oligonucleotide and its complementation district in template: (outer primer/template 3 ' lateral areas)≤(F2c/F2 and R2c/R2)≤(F1c/F1 and R1c/R1).

3. each method, wherein step a i among the claim 1-2) in be RNA as the nucleic acid of template, and step I i) in first nucleic acid synthesize by enzyme with reverse transcriptase activity.

4. the method for amplification of nucleic acid, described nucleic acid have the complementary nucleotide sequence that alternately connects on one bar chain, described method is finished by repeating following steps:

A) provide the step of template, described template comprises the sequence that can form ring in each end region of same chain, when these mutual complementary nucleotide sequences are annealed, form the ring that can carry out base pairing between them, the template that wherein comprises the sequence that forms ring is second nucleic acid that is provided by following step;

I) annealing steps, make the annealing of first oligonucleotide and template F2c district, 3 ' end of this template comprises F2c and is positioned at the F1c district of F2c district 5 ' side, 5 ' end of this template comprises the R1 district and is positioned at the R2 district of R1 district 5 ' side, this first oligonucleotide comprises at least two district F2 and F1c, F1c links to each other with the 5 ' side of F2

F2: have with the district of F2c district complementary nucleotide sequence and

F1c: with the F1c district of F2c district 5 ' side on the template district of basic identical nucleotide sequence is arranged,

The step of ii) synthetic first nucleic acid, described first nucleic acid has and described template complementary nucleotide sequence, described step with the F2 of first oligonucleotide as the synthetic starting point,

Iii) utilize polysaccharase catalysis strand replacement reaction to replace ii) synthetic first nucleic acid in the step, wherein with template in F2c 3 ' side annealed, first outer primer as the synthetic starting point and

Iv) annealing steps makes the ii) R2c district annealing of the nucleic acid of winning of second oligonucleotide and step I, two district R2 and R1c below wherein this second oligonucleotide comprises at least, and R1c links to each other with the 5 ' side of R2,

R2:3 ' end region, its have with R2c district complementary nucleotide sequence and

R1c: with the R1c district of the first nucleic acid R2c district, 5 ' side the district of basic identical nucleotide sequence is arranged,

Then with described second oligonucleotide as synthetic second nucleic acid of synthetic starting point, and utilize polysaccharase catalysis strand replacement reaction to replace this second nucleic acid, wherein with template in 3 ' side annealed, second outer primer of R2c as the synthetic starting point;

B) synthesis step is with steps A) second nucleic acid be synthetic himself the complementary strand of template, being to synthesize starting point with 3 of the above-mentioned template of same chain annealed ' end;

C) annealing steps, making first oligonucleotide and steps A) F2c district in the ring that forms anneals, then carry out synthesis step, with this oligonucleotide is synthetic starting point, synthesize complementary strand by polysaccharase catalysis strand displacement type complementary strand synthesis reaction, with displacement step B) middle synthetic complementary strand, make complementary strand 3 ' end can carry out base pairing, wherein said oligonucleotide comprises and step B) middle 3 ' terminal complementary nucleotide sequence as synthetic starting point; With

D) annealing steps, make at step C) in annealed by metathetical complementary strand 3 ' terminal R1 district and the R1c district in this chain, then carry out synthesis step, with described 3 ' end is synthetic starting point, carrying out complementary strand by polysaccharase catalysis strand displacement type complementary strand synthesis reaction synthesizes, with displacement step C) middle institute synthetic complementary strand, wherein synthetic starting point be same chain 3 ' end can with R1c district annealed R1 district, and, by R1 and R1c annealing, form comprise can carry out base pairing the R2c district at interior ring

5. the method for claim 4, further comprise such step: the template steps A) is, with step C) in oligonucleotide as synthetic starting point institute synthetic complementary strand.

6. claim 4 or 5 method, wherein strand displacement type complementary strand synthesis reaction is to implement under the situation that has the melting temperature(Tm) conditioning agent to exist.

7. the method for claim 6, wherein the melting temperature(Tm) conditioning agent is a trimethyl-glycine.

8. the method for claim 7, wherein the concentration of trimethyl-glycine is 0.2-3.0M in the reaction soln.

9. the test kit of nucleic acid, described nucleic acid has the complementary strand that alternately connects on one bar chain, described complementary nucleotide sequence comprises 5 ' terminal F2 district and is positioned at the F1 district and 5 ' the terminal R2 district of this F2 district 3 ' side and is positioned at the R1 district of this R2 district 3 ' side, and described test kit comprises following component:

I) oligonucleotide, two district F2 and F1c below it comprises at least, F1c links to each other with the 5 ' side of F2,

F2: have with the district of F2c district complementary nucleotide sequence and

F1c: with complementary district, F1 district;

Ii) oligonucleotide, two district R2 and R1c below it comprises at least, and R1c links to each other with the 5 ' side of R2,

R2:3 ' end region, its have with R2c district complementary nucleotide sequence and

R1c: with complementary district, R1 district;

Iii) oligonucleotide, it has and F3c district complementary nucleotide sequence as F2c district 3 ' side in the nucleic acid of template;

Iv) the archaeal dna polymerase of catalysis strand displacement type complementary strand synthesis reaction and,

V) Nucleotide, it is as component substrate iv).

10. the described test kit of claim 9 further comprises following component:

Vi) oligonucleotide, it has and the R3c district complementary nucleotide sequence of R2c district 3 ' side arbitrarily, described any R2c district is arranged in component i) described oligonucleotide is as starting point institute synthetic complementary strand.

11. a test kit that is used to detect target nucleotide sequences wherein on the basis of the described test kit of claim 9, also comprises the detection reagent that is used to detect nucleic acid building-up reactions product.

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