CN1186457C - Homogeneous genetic matrix - Google Patents
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- CN1186457C CN1186457C CNB011265787A CN01126578A CN1186457C CN 1186457 C CN1186457 C CN 1186457C CN B011265787 A CNB011265787 A CN B011265787A CN 01126578 A CN01126578 A CN 01126578A CN 1186457 C CN1186457 C CN 1186457C
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Abstract
The present invention provides a homogeneous phase gene matrix which comprises a substrate, wherein the substrate is provided with 4 to 100, 000 reaction holes, and at least one of the reaction holes is provided with an artificial sequence template primer set. The primer set comprises an oligonucleotide primer pair which is bound specifically on a nucleotide sequence to be detected and is used for initiating the amplification reaction of target nucleic acid, wherein at least one primer is an artificial sequence template primer which comprises a specific binding area (a) and a common area (b). The present invention also provides a method for detecting nucleic acid and/or determining the quantity of the nucleic acid by using the gene matrix. The present invention is easy in use, sensitive and accurate, and can detect and/or determine the quantity in high flux of pathogen in a sample, and the single nucleotide polymorphism, the gene expression, etc. of a genome.
Description
Technical field
The present invention relates to the detection of nucleic acids field, more specifically, relate to the homogeneous genetic matrix that a kind of high-throughput nucleic acid detects usefulness, and use this genetic matrix to detect and/or the method for quantitative nucleic acid.
Background technology
Recently, detect rapidly and accurately and/or quantitative pathogenic agent (as virus, bacterium, fungi) in order to satisfy, and the specific nucleic acid sequence in the normal and undesired gene, have a large amount of technology of having developed.These technology detect and quantitative food, environmental sample, breeding stock and other types material in wide purposes is arranged aspect the microorganism, under these occasions, need to monitor certain and infer microorganism and whether exist.Other application comprise aspects such as being used for medical jurisprudence, molecular pathology, anthropology, archeology and biology.
A kind of common way that realizes this generic task is a nucleic acid hybridization.This method forms the ability of duplex structure based on two nucleic acid chains under conditions suitable, thereby wherein these two nucleic acid chains contain complementation or basic complementary sequence combination specifically.In order to detect and/or quantitative specific nucleotide sequence (being called " target sequence "), need the oligonucleotide (" probe ") of preparation mark, this probe contains the sequence with target complement sequence.In order to detect delicately and/or quantitatively micro-genetic material, many more proven technique have been developed, they are usually directed to amplifying target nucleic acid in specimen (DNA or RNA) and detect subsequently, comprising polymerase chain reaction (PCR), ligase chain reaction (LCR), chain replace amplification (SDA), transcriptive intermediate amplification (transcription mediated amplication, TMA) and self-sustained synthetic reaction (3SR).
Although all these technology all are to detect and differentiate the favourable instrument of micro-target nucleic acid in the sample, they have various problem, and these problems have limited their applicabilitys when being used for routine operation under the clinical experiment room environmental.One of the most difficult problem is that amplifying target nucleic acid in every kind of test is different so that detect with the condition of quantitative analysis subsequently.In other words, be not beneficial to the unified condition of testing standardization.
In addition, nucleic acid detection technique moves towards many targets from single nucleic acid hybridization and PCR reaction and detects simultaneously, and then develops on a fritter substrate and detect thousands of nucleotide sequence simultaneously, i.e. genetic matrix technology (microarray).The genetic matrix technology is a revolution of human sciences's history, has really realized the high throughput testing of nucleic acid, has promoted human genome and back Study on Genome and exploitation, plays a great role in fundamental research and biomedical practice.
The genetic matrix technology is to utilize microelectronics, micromechanics, chemistry, physics and computer technology in microfluid system and unit that the solid chip surface makes up, with discontinuous analytic process related in the life science (as specimen preparation, chemical reaction and analyzing and testing) serialization, integrated and microminiaturized.The genetic matrix technology is of many uses at numerous areas such as drug development, new gene discovery, disease genesis mechanism, medicals diagnosis on disease.Its ultimate principle still is a nucleic acid hybridization.It is fixed on a large amount of probe molecules on the upholder, hybridizes with the sample of mark then, analyzes by detecting intensity of hybridization signal and distribution.
At present, the genetic matrix technology of international popular has the synthetic and two kinds of methods of point sample of original position.
In-situ synthesis comprises that mainly light instructs parallel synthesis (Affymetrix Inc.), microfluidic channel method and molecular seal method.Wherein the light of Affymetrix Inc. instructs parallel synthesis the most ripe; its detailed process is as follows: mercury light shines on the sheet glass of optical masking agent (M1) protection selectively; removing the photosensitive group (X) on the sheet glass, thereby activate the building-up process of DNA.In a light protection of the privileged site coupling of removing photosensitive group base (A-X).Again second optical masking agent (M2) placed on this base that is subjected to the light protection.Constantly remove to protect the oligonucleotide fragment that just can obtain 30 base length with coupling.Many so not homotactic fragments just constitute the DNA square formation.
The point sample method prepares cDNA (or oligonucleotide) probe library at first according to a conventional method, then by special syringe needle or mini sprinkler, respectively different probe solutions, pointwise is distributed on the different loci of glass, nylon or other solid phase substrate surfaces, and makes it to be fixed in corresponding site by the combination of physics and chemistry.Probe fragment except oligonucleotide probe, also can use long gene fragment and nucleic acid analog probe (as PNA) from number of ways.The preparation method of probe can be with conventional dna probe synthetic method, or the cDNA of pcr amplification, EST library etc.
Therefore, the process of genetic matrix technology is as follows: oligonucleotide synthesizes or PCR prepares target gene, and point sample is fixed on the substrate.Simultaneously prepare fluorescent probe, and hybridize, carry out check and analysis subsequently with the substrate that point sample fixes according to different purposes.
Yet, genetic matrix technical development and imperfection, the following defectives of technology itself:
(1) the oligonucleotide combined coefficient of in-situ synthesis is low; The point sample method needs thousands of PCR reaction amplifications to be used for the nucleic acid of point sample, and needs repeatedly repeated authentication;
(2) preparation of sample and marking operation are comparatively loaded down with trivial details, need to extract enough mRNA and can carry out significant notation;
(3) on same substrate, can't look after on the thousands of sampling points and take different hybridization conditions because of the difference of fixed nucleic acid;
(4) cost height, the instrument costliness that needs has limited the popularization and application of this technology;
(5) though according to the size of the strong and weak decision signal amount of hybridization back signal, such is quantitatively very superficial, can not stdn;
(6) cost an arm and a leg.
Therefore, this area press for exploitation easy-to-use, sensitive, accurately detect and/or quantitative sample in the biochip technology of pathogenic agent and genetic expression.
Summary of the invention
Purpose of the present invention just provides and a kind ofly can be used for detecting and/or quantitatively easy-to-use, sensitive, the genetic matrix technology accurately of pathogenic agent and genetic expression in the sample.New technology of the present invention can overcome many limitations of the prior art.
In a first aspect of the present invention, a kind of homogeneous genetic matrix is provided, it comprises:
One substrate has 4-100 on the described substrate, 000 reacting hole;
In at least one described reacting hole, has artificial sequence template primer collection, described primer collection comprises: it is right that specificity is incorporated into detected nucleotide sequence and causes the Oligonucleolide primers of purpose nucleic acid amplification reaction, wherein at least one primer is an artificial sequence template primer, and this artificial sequence template primer comprises:
(a) specific combination district, this specific combination district is positioned at artificial sequence template primer 3 ' end, is used for combining with detected nucleotide sequence specificity;
(b) public domain, this public domain is positioned at 5 of artificial sequence template primer ' hold and has reporter molecules land and public guiding region, but the reporter molecules specificity is incorporated into the complementary strand of this report molecule land or this report molecule land, and the state difference of described reporter molecules under following two kinds of situations, thereby produce detectable signal: (i) be incorporated into the complementary strand of reporter molecules land or this report molecule land and (ii) reporter molecules be cut off or be substituted.
In a preference, also be provided with the check plot on the described substrate, there is at least one reacting hole described check plot, is used to place contrast agents.
In another preference, described substrate is 96 orifice plates or fibre bundle, and described fibre bundle is made up of the respond optical fiber in pond of end.Preferably, described homogeneous genetic matrix also comprises a closure member, is used for the reacting hole of closed substrate.
In a preference, described reporter molecules comprises: FRET (fluorescence resonance energy transfer) type signal probe and contain the oligonucleotide chain of rare earth element.More preferably, described FRET (fluorescence resonance energy transfer) type signal probe is selected from down group: Taqman probe, the two probes of FRET, molecular beacon and PNA signal probe, and between specific combination district and reporter molecules land, there is transcribed spacer, and/or between public guiding region and reporter molecules land, having transcribed spacer, described transcribed spacer is 1-20bp.
In another preference, described artificial sequence template primer collection also comprises the amplification template primer that is used to increase detected nucleic acid-templated quantity.
In another preference, also there is transcribed spacer in artificial sequence template primer between specific combination district and reporter molecules land, and/or between public guiding region and reporter molecules land, having transcribed spacer, described transcribed spacer is 1-20bp, preferably is 5-10bp.
In another preference, the length of the public guiding region of described artificial sequence template primer is 0bp.
In a second aspect of the present invention, passed through a kind of nucleic acid detection method, it comprises step:
(1) sample is added to a substrate, have 4-100 on the described substrate, 000 reacting hole; In at least one described reacting hole, has artificial sequence template primer collection, described primer collection comprises: it is right that specificity is incorporated into detected nucleotide sequence and causes the Oligonucleolide primers of purpose nucleic acid amplification reaction, wherein at least one primer is an artificial sequence template primer, and this artificial sequence template primer comprises:
(a) specific combination district, this specific combination district is positioned at artificial sequence template primer 3 ' end, is used for combining with detected nucleotide sequence specificity;
(b) public domain, this public domain is positioned at 5 of artificial sequence template primer ' hold and has reporter molecules land and public guiding region, but the reporter molecules specificity is incorporated into the complementary strand of this report molecule land or this report molecule land, and the state difference of described reporter molecules under following two kinds of situations, thereby produce detectable signal: (i) be incorporated into the complementary strand of reporter molecules land or this report molecule land and (ii) reporter molecules be cut off or be substituted.
(2) under the specificity condition, carry out nucleic acid amplification reaction,
(3) detectable signal that produced of examining report molecule.
In a preference, use two or more different artificial sequence template primer collection, these artificial sequence template primer collections are incorporated into the different zones of identical target sequence or its combination of the different target sequences of the target sequence of different detected bodies, same detected body, same detected body respectively specifically, and the reporter molecules land of these artificial sequence template primer collections combines with identical or different reporter molecules generation specificity respectively.
Description of drawings
Fig. 1 has shown two kinds of structural representations of artificial sequence template primer of the present invention.
Fig. 2 has shown the various array configurations of artificial sequence template primer collection of the present invention.
Fig. 3 has shown that signal produces several synoptic diagram in the artificial sequence template detection of the present invention.
Fig. 4 is the synoptic diagram of a kind of nucleic acid detection method of the present invention, and use therein artificial sequence template primer collection contains an artificial sequence template primer, its reporter molecules in conjunction with the reporter molecules land of artificial sequence template.
Fig. 5 is the synoptic diagram of a kind of nucleic acid detection method of the present invention, use therein artificial sequence template primer collection contains an artificial sequence template primer, a conventional primer, a public primer and an amplification template primer, its reporter molecules in conjunction with the reporter molecules land of artificial sequence template.
Fig. 6 is the schema of homogeneous genetic matrix detection method of the present invention.
Embodiment
Homogeneous genetic matrix technology of the present invention is the product that the artificial sequence template nucleic acid detection technique combines with fluorescent signal detection in real time and little application of sample technology.In brief, the homogeneous genetic matrix technology may further comprise the steps: utilize related software (as Primer etc.), seek suitable primer fragment on nucleic acid target sequence, design and synthesize satisfactory artificial sequence template primer collection again; Artificial sequence template primer collection and test sample are added to for example 96 orifice plates or more high-density base board; Under same or essentially identical PCR reaction conditions, increase and detect, thereby realize the augmentation detection of a large amount of nucleic acid target sequences.
Most crucial technology is artificial sequence template nucleic acid detection technique (seeing for details hereinafter) in the homogeneous genetic matrix technology of the present invention.As for the other technologies that in homogeneous genetic matrix, adopt, be prior art as microflow control technique, little application of sample technology, photosignal technology, computer technology and bioinformatics technique etc.
As used herein, following word/term has following meanings, unless otherwise indicated.
" nucleic acid ": the oligonucleotide analogs of polynucleotide analogue, RNA or the DNA of Yeast Nucleic Acid (RNA), thymus nucleic acid (DNA), RNA or DNA.
" template ": can be by the total length of the nucleic acid molecule of nucleic acid polymerization enzymatic amplification or partial sequence.Template can be RNA or DNA or its analogue, and can be strand, two strands or partially double stranded.
" artificial sequence template (AT) primer ": artificial sequence template primer is the synthetic oligonucleotide sequence.Referring to Figure 1A, 3 of artificial sequence template primer ' end has the specific combination district, and this specific combination district is complementary to target sequence and extends in amplified reaction as primer.In addition, artificial sequence template primer also contains public domain, and this public domain is positioned at 5 of artificial sequence template primer ' hold and has reporter molecules land (or its complementary sequence) and public guiding region.But the reporter molecules specificity is incorporated into the complementary strand of this report molecule land or this report molecule land.Artificial sequence template primer can be synthetic with the whole bag of tricks well known by persons skilled in the art.
A kind of length of public guiding region of artificial sequence template primer of special shape is 0bp, shown in Figure 1B.
As used herein, " reporter molecules land " refer to artificial sequence template primer on reporter molecules generation bonded zone.Certainly, reporter molecules can directly be incorporated into this report molecule land on the artificial sequence template primer, also can be incorporated into the complementary strand (for simplicity, the zone the same with the reporter molecules sequence on the artificial sequence template primer still is called as " reporter molecules land ") of this land.
In addition, also there is transcribed spacer in a kind of preferred artificial sequence template primer between specific combination district and reporter molecules land, and/or between public guiding region and reporter molecules land, having transcribed spacer, described transcribed spacer is 1-20bp (preferably being 3-10bp, more preferably is 5-10bp).The main purpose of adding transcribed spacer is: (1) regulates the Tm of artificial sequence template primer, makes the Tm of each primer in the artificial sequence template primer collection approaching or identical; (2) prevent the formation of some secondary structure (as hairpin structure, dimer etc.); And/or (3) prevent sterically hindered.Yet should be understood that all needs transcribed spacer under not all situation, do not need transcribed spacer sometimes.Depend on sequence to be detected, those skilled in the art can determine whether artificial sequence template primer needs transcribed spacer, and the length of transcribed spacer and composition.
" reporter molecules " is a kind of molecule that is used to produce detectable signal.The state difference of described reporter molecules under following two kinds of situations, thus produce detectable signal: (i) be incorporated into reporter molecules land or its complementary sequence zone and (ii) reporter molecules be cut off or be substituted.Suitable reporter molecules example is based on the probe of FRET (fluorescence resonance energy transfer) (FRET) principle and contains the oligonucleotide chain of rare earth element.
FRET (fluorescence resonance energy transfer) (FRET): be the interaction that utilizes between different fluorescent substance excitation wavelengths, wavelength of transmitted light, a kind of signal detection principle that some special wavelength light is detected.The probe that produces signal based on this principle is called as FRET (fluorescence resonance energy transfer) type probe.Specially suitable FRET (fluorescence resonance energy transfer) type probe comprises (but being not limited to): Taqman probe, molecular beacon (molecular beacon) (Fig. 3 D), the two probes (Fig. 2 J) of FRET and peptide nucleic acid(PNA) (peptidenucleic acid) signal probe (abbreviating the PNA signal probe as).
" Taqman probe " be a kind ofly have reporter group and 3 at its 5 ' end ' end has the oligonucleotide of quencher group, described quencher group can suppress reporter group and produce detectable signal (for example fluorescence).The Taqman probe design is a prior art, and can obtain (Heid CA, Stevens J, Livak KJ, Williams PM for information about from open approach; Real Time Quantitaive PCR, Genome Res.1996 Oct.; 6 (10): 986-94).
Fig. 3 has shown that signal produces several synoptic diagram in the artificial sequence template detection of the present invention.Fig. 3 A and Fig. 3 B are that the Taqman probe produces detectable signal because of cutting, and Fig. 3 C is that the two probes of FRET are cut because of enzyme or replaced and produce detectable signal.Fig. 3 D is that molecular beacon produces detectable signal because of hybridization.
" artificial sequence template " refers in PCR process of the present invention, amplification nucleotide sequence or its anti sense nucleotide sequence that produce, that mixed artificial sequence template primer.These artificial sequence templates had both had the nucleotide sequence corresponding to target sequence, had again corresponding to the artificial sequence template primer public domain nucleotide sequence of (comprising reporter molecules land, public guiding region and transcribed spacer).In pcr amplification circulation subsequently, these artificial sequence templates work as template.
The method disclosed in the present is classified as the amplification of signal method in principle.Its key has been to use the template (this be called " artificial sequence template ") of the suitable nucleotide sequence that contains artificial sequence as amplification of signal, and therefore corresponding method is called as " foranalysis of nucleic acids of artificial sequence template amplification ".This method can sensitive, accurate and detection of stdn ground and/or quantifying target nucleic acid molecule.
In the present invention, for testing sample without limits, as long as determinand is a nucleic acid molecule.Representational testing sample comprises (but being not limited to): DNA sample, RNA sample and the cDNA sample that obtains through reverse transcription from RNA, and the polynucleotide of other forms of modified.
In artificial sequence template primer of the present invention, the length in described specific combination district is not particularly limited, its length is 6-35bp usually, preferably is 15-25bp.For also being not particularly limited of described public domain, its length is 8-100bp usually, preferably is 20-60bp, more preferably is about 30-50bp.
The Nucleotide that contains in the artificial sequence template primer is selected from A, T, C, G usually.Yet, in described artificial sequence template primer, contain some other Nucleotide producing special detection effect, as increasing specificity, mixing fluorescence molecule or increase and combining of template etc.Suitable example but be not limited to is selected from down the Nucleotide of group: isoG, isoC, 2 '-O-methyl-G, 2 '-O-methyl-C, antigen molecule or biotin labeled Nucleotide and combination thereof.
In reaction system of the present invention, the quantitative relation of reporter molecules and artificial sequence template primer is not particularly limited.Yet preferably, the quantity of described reporter molecules should be more than or equal to the quantity of artificial sequence template primer, and reporter molecules and the ratio of artificial sequence template primer are greater than 1 usually: 1-10: 1, and more preferably be 1.5: 1-5: 1.Like this, in reaction system, artificial sequence template primer just all is in and reporter molecules bonded state basically.
When reporter molecules was the Taqman probe, the Tm of Taqman probe and artificial sequence template primer reporter molecules land should be higher than the Tm of artificial sequence template primer specific combination district and template.Usually exceed 2-15 ℃, preferably exceed 5-12 ℃.
In addition, in artificial sequence template primer collection of the present invention, also can comprise one or more " amplification template primers ".As used herein, " amplification template primer " refers to be used to increase the primer of detected nucleotide sequence (template) quantity.The amplification template primer is incorporated into the upstream or the downstream of sequence to be detected, can improve the quantity of template effectively in amplified reaction, thereby the sensitivity of detection is provided.Should be understood that " amplification template primer " is conventional primer, just its effect is the quantity that improves template to be detected.
In the present invention, as shown in Figure 2, artificial sequence template primer collection has multiple array configuration, comprising (but being not limited to):
(1) downstream is an artificial sequence template primer, and the upstream is conventional primer, and reporter molecules combines (Fig. 2 A) with artificial sequence template primer, or the upstream and downstream primer location exchanges (Fig. 2 B);
(2) primer collection of Fig. 2 A and the combination (Fig. 2 C) that the amplification template primer forms, the primer collection of Fig. 2 B and the combination (Fig. 2 D) that the amplification template primer forms.Be that upstream and downstream is that conventional primer is right, the centre is an artificial sequence template primer, and reporter molecules combines with artificial sequence template primer.
Article (3) two, artificial sequence template primer and two combinations (Fig. 2 E) that the amplification template primer constitutes.Be that upstream and downstream is that conventional primer is right, the centre is the relative artificial sequence template primer of a pair of direction, and reporter molecules combines with artificial sequence template primer;
(4) upstream and downstream is that conventional primer is right, and middle two artificial sequence template primer directions are opposite, and reporter molecules combines (Fig. 2 F) with artificial sequence template primer;
(5) downstream is an artificial sequence template primer, and the upstream is conventional primer, and reporter molecules combines (Fig. 2 G) with the complementary sequence of artificial sequence template primer, or the upstream and downstream primer location exchanges (not shown);
(6) primer collection of Fig. 2 G and the combination (Fig. 2 H) that the amplification template primer forms, be that upstream and downstream is that conventional primer is right, the centre is an artificial sequence template primer, and reporter molecules combines with the artificial sequence template primer complementary sequence, or the artificial sequence template primer direction transformation;
(7) upstream is conventional primer, and the downstream is an artificial sequence template primer, and reporter molecules is marked at (Fig. 2 I) on artificial sequence template primer and the reporter molecules respectively, or artificial sequence template primer direction transformation (not shown); The perhaps primer collection of Fig. 2 I and combination (Fig. 2 J) that the amplification template primer forms;
(8) combination of Fig. 2 E+ Fig. 2 G, promptly upstream and downstream is that conventional primer is right, and the centre is a pair of oppositely relative artificial sequence template primer, and reporter molecules combines (not shown) with the artificial sequence template primer complementary sequence
(9) combination of Fig. 2 F+ Fig. 2 G, promptly upstream and downstream is that conventional primer is right, and middle two artificial sequence template primer directions are opposite, and reporter molecules combines (not shown) with the artificial sequence template primer complementary sequence;
(10) combination of Fig. 2 E+ Fig. 2 I, promptly upstream and downstream is that conventional primer is right, and the centre is a pair of opposite relative artificial sequence template primer, and reporter molecules is marked at (not shown) on artificial sequence template primer and the reporter molecules respectively;
(11) combination of Fig. 2 F+ Fig. 2 I, promptly upstream and downstream is that conventional primer is right, and the centre is two artificial sequence template primers that direction is opposite, and reporter molecules is marked at (not shown) on artificial sequence template primer and the reporter molecules respectively.
In the methods of the invention, on same detected nucleotide sequence, can place one or more artificial sequence template primer collections simultaneously.Can detect the different positions on the detected nucleotide sequence so simultaneously.
In the present invention, for for artificial sequence template primer bonded reporter molecules, different artificial sequence template primers can be in conjunction with identical reporter molecules, also can be in conjunction with different reporter molecules (for example have send out different the reporter group of fluorescence and the reporter molecules of corresponding quencher group, the reporter molecules that sequence is identical or different).
The length of the artificial sequence template that amplifies for artificial sequence template primer collection is not particularly limited.Apart the distance expression on template of 3 ends in specific combination district of two primers of pressing artificial sequence template primer collection is generally 1bp-10kb, preferably is 1-2kb, more preferably is 1-500b, is 1-100bp best.Especially be when spacing during, can design at for example primer collection of pathogenic agent high conserved region, thereby reduce false negative rate less than 100bp.In addition, spacing is short more, can bring into play the general character of artificial sequence template more.
Further specify the present invention below in conjunction with accompanying drawing.
The primer collection (combination shown in Fig. 2 A) that amplification pattern (), use contain artificial sequence template primer and conventional primer carries out the nucleic acid amplification analysis
Now referring to Fig. 4.In this example, use an artificial sequence template primer collection, this primer collection comprises an artificial sequence template primer 1 and a conventional primer 2.In addition, comprise also in the reaction system that public primer 3 and reporter molecules 4 constitute.In this example, sample to be detected is RNA or DNA.
Step 1: primer collection and testing sample are placed reaction system.
Step 2: under appropriate condition, anneal (or hybridization), promptly the specific combination district of 3 of artificial sequence template primer 1 ' end is incorporated into target RNA or dna sequence dna.
Step 3: at reverse transcriptase (for the RNA target sequence) or archaeal dna polymerase (for the DNA target sequence), 3 of artificial sequence template primer ' end extends to target sequence 5 ' end, forms RNA/DNA or DNA/DNA two strands.
Step 4: the sex change under appropriate condition of the double-strandednucleic acid of formation forms strand target sequence and new synthetic dna sequence dna (being artificial sequence template).Perhaps use RNase hydrolysis RNA chain, form new synthetic dna sequence dna (being artificial sequence template).
In case of necessity, under appropriate condition, repeat above-mentioned steps 3-4.
Step 5: conventional primer 2 is incorporated into the complementary region of new synthetic dna sequence dna.
Step 6: in the presence of archaeal dna polymerase, 3 of conventional primer 2 ' end extends to artificial sequence template sequence 5 ' end, double-stranded DNA (new synthetic dna sequence dna also is an artificial sequence template in this two strands).When 3 of conventional primer 2 ' end extended to the reporter molecules place that is incorporated into the reporter molecules land, following 3 kinds of situations can take place:
(1) extension stops;
(2) continue to extend, reporter molecules is replaced from artificial sequence template;
(3) continue to extend, and cut off or destroy reporter molecules.
Under first and second kinds of situations, reporter molecules is still complete, therefore can not produce detectable signal.And under the third situation, because the quencher group on reporter molecules (as the Taqman probe) 5 ' end is by 5 ' nuclease institute hydrolysis of archaeal dna polymerase (as the Taq polysaccharase), therefore at the reporter group at Taqman probe 3 ' end place just no longer by quencher, thereby cause producing detectable signal (as fluorescent signal).
Step 7: in next circulation, during annealing after sex change, three kinds of situations can take place:
7a: artificial sequence template primer 1 is incorporated into the new synthetic dna sequence dna that contains conventional primer 2 sequence.
7b: public primer 3 is incorporated into the new synthetic dna sequence dna that contains conventional primer 2 sequence.
7c: conventional primer 2 is in conjunction with the artificial sequence template (being similar to step 5, not shown) that contains artificial sequence primer 1 sequence.
Step 8: divide three kinds of situations:
8a: artificial sequence template primer 1 extends to 5 of the DNA chain that contains conventional primer 2 sequence ' end, forms the dna double chain.In the next round circulation, these dna double chains are exactly new artificial sequence template.
8b: public primer 3 extends to 5 of the DNA chain that contains conventional primer 2 sequence ' end, forms the dna double chain.In the next round circulation, these dna double chains are exactly new artificial sequence template.
8c:, when 3 of conventional primer 2 ' end extends to the reporter molecules place that is incorporated into the reporter molecules land, following 3 kinds of situations can take place with identical in the step 6:
(1) extension stops;
(2) continue to extend, reporter molecules is replaced from artificial sequence template;
(3) continue to extend, and cut off or destroy reporter molecules.
Under first and second kinds of situations, reporter molecules is still complete, therefore can not produce detectable signal.And under the third situation, because the quencher group on reporter molecules (as the Taqman probe) 5 ' end is by 5 ' nuclease institute hydrolysis of archaeal dna polymerase (as the Taq polysaccharase), therefore at the reporter group at Taqman probe 3 ' end place just no longer by quencher, thereby cause producing detectable signal (as fluorescent signal).
Step 9: repeating step 7 and 8.
Through after some circulations, identical with the PCR principle, also be that exponentially level (or being similar to exponential relationship) increases because of reporter molecules is cut off the detectable signal that produces.At detectable signal is under the situation of fluorescence, and these signals can be used the real-time fluorescence reading apparatus, and as Roche ' s LightCycler, perhaps ABI GeneAmp 5700 or GeneAmp 7700 etc. carry out The real time measure; Also can use static fluorescence reading apparatus, after nucleic acid amplification reaction finishes, carry out fluorometric assay.
Artificial sequence template primer collection shown in amplification pattern (two), the use Fig. 2 C carries out the nucleic acid amplification analysis
Now referring to Fig. 5.In this example, use an artificial sequence template primer 1, conventional primer 2 and amplification template primer 5 are formed a primer collection.Also comprise public primer 3 and reporter molecules 4 in the reaction system.In this example, sample to be detected is DNA or RNA.
Step 1: primer collection and testing sample are placed reaction system.
Step 2: under appropriate condition, anneal (or hybridization), two kinds of situations take place this moment:
2a: the specific combination district of 3 of artificial sequence template primer 1 ' end is incorporated into target RNA or dna sequence dna.
2b: amplification template primer 5 combines with target RNA or dna sequence dna.
Step 3:
3a: corresponding to above-mentioned first kind of situation, under reverse transcriptase (for the RNA target sequence) or archaeal dna polymerase (for the DNA target sequence) effect, 3 of artificial sequence template primer 1 ' end extends to target sequence 5 ' end, forms RNA/DNA or DNA/DNA two strands.
3b: corresponding to above-mentioned second kind of situation, under reverse transcriptase (for the RNA target sequence) or archaeal dna polymerase (for the DNA target sequence) effect, 3 of amplification template primer 5 ' end extends to target sequence 5 ' end, forms RNA/DNA or DNA/DNA two strands.
Step 4: the sex change under appropriate condition of the double-strandednucleic acid of formation forms strand target sequence and new synthetic dna sequence dna (being artificial sequence template).Perhaps use RNase hydrolysis RNA chain, form new synthetic dna sequence dna and (under first kind of situation, form artificial sequence template; Under second kind of situation, provide and conventional primer 2 bonded nucleic acid sequence).
In case of necessity, repeating step 2,3 and 4 (choosing wantonly) under appropriate condition.
Step 5: in next circulation, during annealing after sex change, four kinds of situations can take place:
5a: conventional primer 2 is incorporated into the DNA chain that new synthetic contains artificial sequence template primer 1.
5b: conventional primer 2 is incorporated into the DNA chain that new synthetic contains amplification template primer 5.
5c: the specific combination district of 3 of artificial sequence template primer 1 ' end is incorporated into target RNA or dna sequence dna (with step 2a, not shown).
5d: amplification template primer 5 combines (with step 2b, not shown) with target RNA or dna sequence dna.
Step 6:
6a: corresponding to 5a, in the presence of archaeal dna polymerase, 3 of conventional primer 2 ' end extends to artificial sequence template sequence 5 ' end, double-stranded DNA (new synthetic dna sequence dna also is an artificial sequence template in this two strands).When 3 of artificial sequence template primer 2 ' end extended to the reporter molecules place that is incorporated into the reporter molecules land, following 3 kinds of situations can take place:
(1) extension stops;
(2) continue to extend, reporter molecules replaced from artificial sequence template:
(3) continue to extend, and cut off or destroy reporter molecules.
Under first and second kinds of situations, reporter molecules is still complete, therefore can not produce detectable signal.And under the third situation, because the quencher group on reporter molecules (as the Taqman probe) 5 ' end is by 5 ' nuclease institute hydrolysis of archaeal dna polymerase (as the Taq polysaccharase), therefore at the reporter group at Taqman probe 3 ' end place just no longer by quencher, thereby cause producing detectable signal (as fluorescent signal).
6b: corresponding to 5b, in the presence of archaeal dna polymerase, 3 of conventional primer 2 ' end extends to the nascent strand 5 that contains amplification template primer 5 sequences ' end, forms the dna double chain.In the next round circulation, these dna double chains are exactly new artificial sequence template.
6c and 6d:, form new artificial sequence template (not shown) respectively with step 3a and 3b corresponding to 5c and 5d.
Step 7: in next circulation, during annealing after sex change, four kinds of situations can take place:
7a: conventional primer 2 is incorporated into the DNA chain (same 5a, not shown) that new synthetic contains artificial sequence template primer 1.
7b: conventional primer 2 is incorporated into the DNA chain (same 5b, not shown) that new synthetic contains amplification template primer 5.
7c: the specific combination district of 3 of artificial sequence template primer 1 ' end is incorporated into target RNA or dna sequence dna (being similar to step 2a).
7d: amplification template primer 5 combines (with step 2b, not shown) with target RNA or dna sequence dna;
7e: public primer 3 is incorporated into the new synthetic dna sequence dna that contains conventional primer 2 sequence.
Step 8: in extension process next time, following several situation may take place:
8a: in the presence of archaeal dna polymerase, 3 of conventional primer 2 ' end extends to artificial sequence template sequence 5 ' end, double-stranded DNA (new synthetic dna sequence dna also is an artificial sequence template in this two strands).When 3 of artificial sequence template primer 2 ' end extended to the reporter molecules place that is incorporated into the reporter molecules land, following 3 kinds of situations can take place:
(1) extension stops;
(2) continue to extend, reporter molecules is replaced from artificial sequence template;
(3) continue to extend, and cut off or destroy reporter molecules.
Under first and second kinds of situations, reporter molecules is still complete, therefore can not produce detectable signal.And under the third situation, because the quencher group on reporter molecules (as the Taqman probe) 5 ' end is by 5 ' nuclease institute hydrolysis of archaeal dna polymerase (as the Taq polysaccharase), therefore at the reporter group at Taqman probe 3 ' end place just no longer by quencher, thereby cause producing detectable signal (as fluorescent signal).
8b: same 6b, in the presence of archaeal dna polymerase, 3 of conventional primer 2 ' end extends to the nascent strand 5 that contains amplification template primer 5 sequences ' end, forms the dna double chain.In the next round circulation, these dna double chains are exactly new artificial sequence template.
8c: same 6c forms new artificial sequence template (being similar to step 3a).
8d: same 6d forms new artificial sequence template (being similar to 3b).
8e: public primer 3 extends to 5 of the DNA chain that contains conventional primer 2 sequence ' end, forms the dna double chain.In the next round circulation, these dna double chains are exactly new artificial sequence template.
Step 9: repeating step 7 and 8.
Through after some circulations, identical with the PCR principle, also be that exponentially level (or being similar to exponential relationship) increases because of reporter molecules is cut off the detectable signal that produces.At detectable signal is under the situation of fluorescence, these signals can be used the real-time fluorescence reading apparatus, as Roche ' s LightCycler, perhaps ABI GeneAmp 5700 or GeneAmp 7700 etc. carry out The real time measure: also can use static fluorescence reading apparatus, carry out fluorometric assay after nucleic acid amplification reaction finishes.
In the methods of the invention, reporter molecules also can combine (Fig. 2 G and Fig. 2 H) with the complementary strand of reporter molecules land on the artificial sequence template, perhaps use two artificial sequence templates and two conventional primers (combination of Fig. 2 E and 2F), its amplification process and amplification pattern () and (two) are basic identical
In the present invention, the another kind of mode that produces signal is to use the two probes of FRET, and promptly the report signal group is marked at respectively on artificial sequence template and the reporter molecules.Referring to Fig. 3 C and 3C '.At this moment, artificial sequence template primer collection comprises: an artificial sequence template primer 1 and an a pair of conventional primer 2 that is marked with FRET group such as FAM fluorescein.In reaction system, also comprise reporter molecules 4 (oligonucleotide) with another FRET group such as mark Dabcyl.
Step 1-5, step 1-5 is similar with amplification pattern ().
In step 6: under first kind of situation, in the presence of archaeal dna polymerase, 3 of conventional primer 2 ' end extends to artificial sequence template sequence 5 ' end, double-stranded DNA (new synthetic dna sequence dna also is an artificial sequence template in this two strands).When 3 of artificial sequence template primer 2 ' end extended to the reporter molecules place that is incorporated into the reporter molecules land, following 3 kinds of situations can take place:
(1) extension stops;
(2) continue to extend, reporter molecules is replaced from artificial sequence template;
(3) continue to extend, and cut off or destroy reporter molecules.
Under first situation, reporter molecules is combined in artificial sequence template primer, with the FAM molecule on the Dabcyl cancellation artificial sequence template primer of mark on it, therefore can not produce detectable signal.Under second kind of situation, though reporter molecules is complete,, make the mutual cancellation between reporter molecules and artificial sequence template primer destroyed, thereby produce detectable signal (seeing Fig. 3 C ') because nascent strand has replaced reporter molecules.Under the third situation, the quencher group of reporter molecules is by 5 ' nuclease institute hydrolysis of archaeal dna polymerase (as the Taq polysaccharase), therefore reporter molecules on the artificial sequence template primer such as FAM be just no longer by quencher, thereby cause producing detectable signal (as fluorescent signal) (Fig. 3 C).
Under second kind of situation, in the presence of archaeal dna polymerase, 3 of conventional primer 2 ' end extends to the nascent strand 5 that contains amplification template primer 5 sequences ' end, forms artificial sequence template.
Step 7-9 is similar in step 7-9 and the above-mentioned amplification pattern ().
Through after some circulations, identical with the PCR principle, also be that exponentially level (or being similar to exponential relationship) increases because of reporter molecules is cut off or replaces the detectable signal that produces.At detectable signal is under the situation of fluorescence, and these signals can be used the real-time fluorescence reading apparatus, and as Roche ' s LightCycler, perhaps ABI GeneAmp 5700 or GeneAmp 7700 etc. carry out The real time measure; Also can use static fluorescence reading apparatus, after nucleic acid amplification reaction finishes, carry out fluorometric assay.
Artificial sequence template nucleic acid detection technique of the present invention has the advantage that obviously is better than prior art, and its major advantage comprises:
(1) being easy to combined type detects
Mix this artificial sequence template district and can in new synthetic dna sequence dna, introduce the one section sequence that does not belong to target sequence.And new synthetic dna sequence dna can be used as the template of further DNA cloning.Irrelevant with the number of different target sequences, new synthetic dna sequence dna is that major part is identical-artificial sequence template region sequence, and this makes variant target sequence be transformed into the sequence with denominator.Therefore, can under identical or essentially identical condition, continue to handle or operation, be easy to realize that for example combined type such as multitube identical conditions or the multiple target sequence of a pipe detects this dna sequence dna.
(2) higher sensitivity for analysis
Contain the primer collection of two artificial sequence template primers by use, perhaps the different loci to single target sequence designs a plurality of artificial sequence template primer collections, can produce the higher levels of signal to noise ratio of single probe patterns in the prior art.
In addition, use the amplification template primer also to can further improve detection sensitivity.
(3) high accuracy
Artificial sequence template primer of the present invention and artificial sequence template nucleic acid detection method, can reduce the probability of false negative that is caused by following factors:
(a) in the secondary structure at probe (or primer) the binding site place of target sequence, these secondary structures may influence the combination of probe (or primer) effectively;
(b) in the change of probe (or primer) the binding site place of target sequence sequence, these variations can be because different hypotype or sudden changes causes.For example in HCV,, can cause HCV to undergo mutation or make a variation owing to used various medicines.As using the conventional nucleic acid detection method, regular meeting causes false negative.And find out high conservative short zone (as 100bp or shorter) in the various biologies genetic material of (comprising pathogenic agent) is very easily.Utilize the present invention, can design at these artificial sequence template primer collections short, high conserved region territory (as 40-50bp), thereby reduce false negative.According to, the inventor finds that with many detections to artificial sequence template primer collection HCV mutant strain false negative rate reduces 50%.
(c) fracture of long segment target sequence in sample processing or treating processes, and this fracture occurs in the region intermediate of the amplification of long segment, what can cause that complementary dna sequence duplicates stops.
(4) simplify or eliminate multiple detection
When needs detect multiple pathogenic agent, often need carry out independent detection at present to certain pathogenic agent.This is because the optimum reaction condition of different detection reaction has nothing in common with each other, and therefore, when carrying out nucleic acid amplification reaction in same reaction tubes, the efficient of each amplified reaction is difficult near consistent, thereby is difficult to detect simultaneously in same reaction tubes multiple pathogenic agent.
Use technology of the present invention, because the major part of artificial sequence template is identical, promptly therefore the artificial sequence template district is convenient in the reaction conditions stdn that makes the PCR that detects each pathogenic agent, thereby realizes the detection to multiple pathogenic agent in a pipe.This makes the technology of the present invention occasions such as sample survey that are specially adapted to donate blood.
(5) reduce many times of analysis costs that use the fluorescent mark technology
In the prior art, the fluorescence labeling probe that needs similar number for a plurality of target nucleic acid sequences to be measured.In contrast, can only use a kind of public reporter probe for detecting a plurality of target sequences in the present invention, therefore can reduce cost greatly.
In the present invention, be not particularly limited for substrate.As long as on this substrate several reacting holes (being reaction tank) are arranged.The number in hole is generally 4-100, and 000 preferably is 9-10000, more preferably is 16-5000.The shape and the position of reacting hole are not particularly limited, and can be column, poroid or gear-like, and be recessed or protruding, can fix, also removable.The size of reaction tank is generally diameter 10-8000 micron, preferably is the 20-1000 micron, more preferably about 200 microns: its volume is 1nl-100ul, preferably is 100nl-50ul, more preferably is 500nl-1ul.
Reaction tank is to the variation of ambient temperature sensitivity, and thermal conduction is good, can carry out the PCR reaction fully.Its heating and cooling medium can be various media such as metal, air, water and light.
Substrate can be made by glass, polymkeric substance or pottery.A kind of common substrate is the porous plate of 96 hole forms, and it is normally made with polymer materialss such as polypropylene.
A kind of special substrate is the aggregate of being made by optical fiber.Many optical fiber are assembled an optical fiber aggregate (fibre bundle), utilize the method for chemical corrosion, make an end of every optical fiber form a reaction tank.Like this, optical fiber aggregate has just constituted a matrix that contains a plurality of reaction tanks.The diameter of optical fiber is variable, and its common size is 0.The 1-50 millimeter preferably is the 1-10 millimeter.Optical fiber in each fibre bundle is generally 4-100, and 000, preferably be 9-10000, more preferably be 16-5000.Because optical fiber has good photoconduction function, so the variations in light that produces in the reaction tank is easy to be gathered and analyze, and simplified the detection of homogeneous genetic matrix.
Substrate should have the capping that is complementary with reacting hole, and as film, heat-resisting capping, transparent cover that polymkeric substance is made or paraffin or not volatile, heat-resisting and proportion are less than the organic solvent of water.A kind of preferred capping is incorporate capping, the transparent cover of making as polypropylene.In addition, also can be after having added reaction reagent, one or more polymerisable monomers directly adding to above the reaction reagent, are directly being formed disposable capping by polymerization.
In a preference of the present invention, the bottom of reacting hole or capping are transparent, so just can realize detecting in real time in the PCR reaction process.
In homogeneous genetic matrix of the present invention, a kind of signal acquisition method of simplification is arranged.In the method, optical fiber and capping are integrated design, make the direct insertion reaction of optical fiber pond, the fluorescence that makes the reaction generation is directly by fiber optic conduction entering signal acquisition system.Can make the light conducting capping of plurality of specifications by demand, insert instrument in use on demand, can satisfy the needs that different flux detect.
When actually operating, sample and corresponding primer collection are added to each reacting hole on the substrate.A kind of preferable mode is in advance various primer collections to be added to each reacting hole, and then adds response sample.Then, add upper cover, carry out the PCR reaction, and carry out live signal collection and information processing (Fig. 6) with for example instrument such as PE5700 type, PE7700 type fluorescent PCR instrument.Perhaps, after reaction finishes, use static fluorescence readout instrument or fluorescent scanning instrument to carry out data gathering and processing.
Homogeneous genetic matrix technology of the present invention is the innovation on traditional genetic matrix meaning, and its design and making all are better than traditional genetic matrix.Its function can replace traditional genetic matrix fully, and detect fast, sensitive, special, cost is lower, in some fields such as aspects such as medical diagnosis on disease, SNP detections, gene expression analysis the possibility that replaces traditional genetic matrix is arranged greatly.Particularly, its major advantage is:
1. use difficulty and cost low.Mainly show as:
(1) experimental period very short, only need tens of minutes;
(2) reagent consumption is extremely low;
(3) save a large amount of manpower and materials;
(4) save space and PCR instrument.
2. homogeneous reaction: the speed of response index raises.
3. can adopt the real-time fluorescence detection technique, highly sensitive.
4. detection by quantitative is easy: the quantitative wide ranges of three-dimensional space.
5. detection background is low: no matter use how many reporter molecules to be used for mRNA or DNA cloning, all do not have spontaneous or the generation of background fluorescence signal.Because the reporter group in the reporter molecules is suppressed by quenching group.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.
Embodiment 1
The artificial sequence template primer nucleic acid amplification detects HCV virus (RNA viruses)
In this embodiment, designed the artificial sequence template primer collection that comprises an artificial sequence template primer and a conventional primer, the reaction process part as shown in Figure 4.
In the PCR scheme be: become DNA:RT (50 ℃, 20 minutes, 95 ℃, 5 minutes) from the RNA reverse transcription; PCR:50 ℃, 2 minutes, 94 ℃ 5 minutes; 94 ℃ 20 seconds; With 61 ℃ 40 seconds, totally 40 circulations.
Artificial sequence template primer is:
5-AAGGAACAGG CGGCGACGAA TCAACGACAG AACGCAACCC AACGCTACTC-3′(SEQ ID NO：1)
Conventional primer:
5′-GTGCCCCCGC AAGACT-3′(SEQ ID NO：2)
The Taqman probe sequence that uses is:
5′-TCGTCGCCGC CTGTTCCTTA-3′(SEQ ID NO：3)
Use therein reporter group is 6-Fluoresceincarboxylic acid (6-carboxyfluorescein, 6-FAM) (be positioned at 5 ' end), the quencher group is 6-carboxyl-tetramethyl-rhodamine (6-carboxy-tetramethyl-rhodamine, TAMRA) (is positioned at 3 ' end).
The detecting instrument that uses during detection is ABI GeneAmp 5700, and excitation light source is a halogen lamp, and wavelength is 488nm.
Increase at the ABI5700 gene-amplificative instrament with above-mentioned primer and corresponding Taqman probe.As a result, add different extent of dilution positive and fluorescent signal occurs, and fluorescent signal does not all appear when (Ct＞40) in negative sample and other non-special control samples (sample that contains other pathogenic agent) when amplified reaction finishes in difference circulation (Ct).
Embodiment 2
Artificial sequence template augmentation detection HBV virus (dna virus)
In this embodiment, designed the artificial sequence template primer collection that comprises an artificial sequence template primer and a conventional primer, the reaction process part as shown in Figure 4.
In the PCR scheme be: PCR:50 ℃, 2 minutes, 94 ℃ 5 minutes; 94 ℃ 20 seconds; With 61 ℃ 40 seconds, totally 40 circulations.
Artificial sequence template primer is respectively:
5-AAGGAACAGG CGGCGACGAA TCAACGACAG AACCAGCGAT AGCCA GGACA-3′(SEQ ID NO：4)
Conventional primer:
5′-CCTCCAATCA CTCACCAACC-3′(SEQ ID NO：5)
The Taqman probe sequence that uses is:
5′-TCGTCGCCGC CTGTTCCTTA-3′(SEQ ID NO：3)
Use therein reporter group is 6-Fluoresceincarboxylic acid (6-FAM) (being positioned at 5 ' end), and the quencher group is 6-carboxyl-tetramethyl-rhodamine (TAMRA) (being positioned at 3 ' end).
The detecting instrument that uses during detection is ABI GeneAmp 5700, and excitation light source is a halogen lamp, and wavelength is 488nm.
Increase at the ABI5700 gene-amplificative instrament with above-mentioned primer and corresponding Taqman probe.As a result, add different extent of dilution positive and fluorescent signal occurs, and fluorescent signal does not all appear when (Ct＞40) in negative sample and other non-special control samples (sample that contains other pathogenic agent) when amplified reaction finishes in difference circulation (Ct).
Artificial sequence template augmentation detection HCV virus (RNA viruses)
In this embodiment, designed the artificial sequence template primer collection that comprises an artificial sequence template primer, a conventional primer and a public primer, reaction process as shown in Figure 4.
In the PCR scheme be: become DNA:RT (50 ℃, 20 minutes, 95 ℃, 5 minutes) from the RNA reverse transcription: PCR:50 ℃, 2 minutes, 94 ℃ 5 minutes; 94 ℃ 20 seconds; With 61 ℃ 40 seconds, totally 40 circulations.
Artificial sequence template primer is:
5-TCATCCACAT CCCACCTCAT CAGGAACAGG CGGCGACGAA TCAACGACAG AACGCAACCCAACGCTACTC-3′(SEQ ID NO：6)
Conventional primer:
5′-GTGCCCCCGC AAGACT-3′(SEQ ID NO：2)
The public primer sequence that uses is:
5′-TCATCCACAT CCCACCTCAT-3′(SEQ ID NO：7)
The Taqman probe sequence that uses is:
5′-TCGTCGCCGC CTGTTCCTTA-3′(SEQ ID NO：3)
Use therein reporter group is 6-Fluoresceincarboxylic acid (6-FAM) (being positioned at 5 ' end), and the quencher group is 6-carboxyl-tetramethyl-rhodamine (TAMRA) (being positioned at 3 ' end).
The detecting instrument that uses during detection is ABI GeneAmp 5700, and excitation light source is a halogen lamp, and wavelength is 488nm.
Increase at the ABI5700 gene-amplificative instrament with above-mentioned primer and corresponding Taqman probe.As a result, add different extent of dilution positive and fluorescent signal occurs, and fluorescent signal does not all appear when (Ct＞40) in negative sample and other non-special control samples (sample that contains other pathogenic agent) when amplified reaction finishes in difference circulation (Ct).
Embodiment 4
Artificial sequence template augmentation detection HCV virus (RNA viruses)
In this embodiment, designed the artificial sequence template primer collection that comprises an artificial sequence template primer, a pair of conventional primer and a public primer, reaction process as shown in Figure 5.
In the PCR scheme be: become DNA:RT (50 ℃, 20 minutes, 95 ℃, 5 minutes) from the RNA reverse transcription; PCR:50 ℃, 2 minutes, 94 ℃ 5 minutes; 94 ℃ 20 seconds; With 61 ℃ 40 seconds, totally 40 circulations.
Artificial sequence template primer is:
5-TCATCCACAT CCCACCTCAT CAGGAACAGG CGGCGACGAA TCAACGACAG AACGCAACCCAACGCTACTC-3′(SEQ ID NO：6)
Conventional primer 1:
5′-GTGCCCCCGC AAGACT-3′(SEQ ID NO：2)
Conventional primer 2:
5′-TGAGTGTCGTACAGC CTCCAGG-3′(SEQ ID NO：8)
The public primer sequence that uses is:
5′-TCATCCACAT CCCACCTCAT-3′(SEQ ID NO：7)
The Taqman probe sequence that uses is:
5′-TCGTCGCCGC CTGTTCCTTA-3′(SEQ ID NO：3)
Use therein reporter group is 6-Fluoresceincarboxylic acid (6-FAM) (being positioned at 5 ' end), and the quencher group is 6-carboxyl-tetramethyl-rhodamine (TAMRA) (being positioned at 3 ' end).
The detecting instrument that uses during detection is ABI GeneAmp 5700, and excitation light source is a halogen lamp, and wavelength is 488nm.
Increase at the ABI5700 gene-amplificative instrament with above-mentioned primer and corresponding Taqman probe.As a result, add different extent of dilution positive and fluorescent signal occurs, and fluorescent signal does not all appear when (Ct＞40) in negative sample and other non-special control samples (sample that contains other pathogenic agent) when amplified reaction finishes in difference circulation (Ct).
Artificial sequence template augmentation detection HCV virus (RNA viruses) and HBV virus (dna virus)
In this embodiment, designed the artificial sequence template primer collection that comprises two artificial sequence template primers, two pairs of conventional primers and a public primer, reaction process as shown in Figure 5.
In the PCR scheme be: become DNA:RT (50 ℃, 20 minutes, 95 ℃, 5 minutes) from the RNA reverse transcription; PCR:50 ℃, 2 minutes, 94 ℃ 5 minutes; 94 ℃ 20 seconds; With 61 ℃ 40 seconds, totally 40 circulations.
HCV virus artificial sequence template primer is:
5-TCATCCACAT CCCACCTCAT CAGGAACAGG CGGCGACGAA TCAACGACAG AACGCAACCCAACGCTACTC-3′(SEQ ID NO：6)
HBV virus artificial sequence template primer is:
5′-TCATCCACAT CCCACCTCAT CAGGAACAGG CGGCGACGAA TCATCCAGTC TATGTTTCCCTCTTGTTGCT-3′(SEQ ID NO：9)
The conventional primer 1 of HCV virus:
5′-GTGCCCCCGC AAGACT-3′(SEQ ID NO：2)
The conventional primer 2 of HCV virus:
5′-TGAGTGTCGTACAGC CTCCAGG-3′(SEQ ID NO：8)
The conventional primer 1 of HBV virus:
5′-CCTCCAATCA CTCACCAACC-3′(SEQ ID NO：5)
The conventional primer 2 of HBV virus:
5′-AGTTTCCGTC CGAAGGTTT-3′(SEQ ID NO：10)
The public primer sequence that uses is:
5′-TCATCCACAT CCCACCTCAT-3′(SEQ ID NO：7)
The Taqman probe sequence that uses is:
5′-TCGTCGCCGC CTGTTCCTTA-3′(SEQ ID NO：3)
Use therein reporter group is 6-Fluoresceincarboxylic acid (6-FAM) (being positioned at 5 ' end), and the quencher group is 6-carboxyl-tetramethyl-rhodamine (TAMRA) (being positioned at 3 ' end).
The detecting instrument that uses during detection is ABI GeneAmp 5700, and excitation light source is a halogen lamp, and wavelength is 488nm.
Increase at the ABI5700 gene-amplificative instrament with above-mentioned primer and corresponding Taqman probe.As a result, add different extent of dilution HCV with (or) the HBV positive fluorescent signal occurs in different circulations (Ct), contains HBV simultaneously and the HCV positive does not influence detection sensitivity, its Ct value is mainly determined by high concentration virus.And fluorescent signal does not all appear when (Ct＞40) in negative sample and other non-special control samples (sample that contains other pathogenic agent) when amplified reaction finishes.
Embodiment 6
Artificial sequence template augmentation detection HCV virus (RNA viruses)
In this embodiment, designed the artificial sequence template primer collection that comprises a fluorescein-labeled artificial sequence template primer and a pair of conventional primer, the reaction process part as shown in Figure 8.
In the PCR scheme be: become DNA:RT (50 ℃, 20 minutes, 95 ℃, 5 minutes) from the RNA reverse transcription; PCR:50 ℃, 2 minutes, 94 ℃ 5 minutes; 94 ℃ 20 seconds; With 61 ℃ 40 seconds, totally 40 circulations.
The artificial sequence template primer sequence is:
5′-AAGGAACAGG CGGCGACGAA TCAACGACAG AACGCAACCC AACGCTACTC-3′(SEQ ID NO：11)
Use therein reporter group is 6-Fluoresceincarboxylic acid (6-FAM) (being positioned at 5 ' end)
Conventional primer:
5′-GTGCCCCCGC AAGACT-3′(SEQ ID NO：2)
The amplification template primer:
5′-TGAGTGTCGTACAGC CTCCAGG-3′(SEQ ID NO：8)
The reporter probe sequence of using is:
5′-TCGTCGCCGC CTGTTCCTTA-3′(SEQ ID NO：3)
Use therein quencher group is Dabcyl (i.e. four (4-methylamino phenyl azo)-phenylformic acid) (being positioned at 3 ' end).
The detecting instrument that uses during detection is ABI GeneAmp 5700, and excitation light source is a halogen lamp, and wavelength is 488nm.
Increase at the ABI5700 gene-amplificative instrament with above-mentioned primer and corresponding Taqman probe.As a result, if only use conventional primer 1 and artificial sequence template primer to increase, compare with the artificial sequence template primer collection of amplification template primer with adding conventional primer simultaneously, its detection sensitivity can differ 10～100 times.Add different extent of dilution positive and fluorescent signal occurs, and fluorescent signal does not all appear when (Ct＞40) in negative sample and other non-special control samples (sample that contains other pathogenic agent) when amplified reaction finishes in difference circulation (Ct).
Embodiment 7
Tubercule bacillus rpoB SNPs detection platform makes up
Tubercule bacillus often causes people and domestic animal generation tuberculosis, is one of current important unmanageable transmissible disease.The normal origination point sudden change of the rpoB gene of tubercule bacillus shows as single nucleotide polymorphism (SNPs).These SNPs are the drug-fast major causes of tuberculosis, and the resistance of tuberculosis is a great problem of its prevention and treatment.Select tuberculosis rpoB gene and detect its SNPs, the resistance from gene level discovery and prediction tuberculosis has important social benefit and economic benefit.
At the SNPs of rpoB gene,, designed tens of to primer according to the requirement of UT reagent technology.These primers contain general probe template sequence and primer and the contrast primer corresponding with the rpoB gene mutation site, and 3 of primer ' end is designed by particular requirement.So the amplified reaction corresponding with the mutational site all carries out on PE5700 fluorescent real time PCR instrument under identical conditions.Whether the result conforms to expection, promptly can discern detected sudden change and take place under same amplification condition, where occurs in, and what kind of sudden change has taken place.
The structure explanation UniArray Matrix Technology of this platform technology is feasible, can be used for extensive, high-throughout SNPs and detects.
1, design of primers (not containing public guiding region)
The mutational site of tubercule bacillus rpoB concentrates on the single nucleotide mutation in sites such as 511,513,516,526,531 of aminoacid sequence.At these site design satisfactory conventional primer of design and artificial sequence template primer.
Conventional primer 1:
5′-CAATCAAGGA GTTCTTCGGC A-3′(SEQ ID NO：12)
Conventional primer 2:
5′-GGCACGCTCA AGTGACAGAC-3′(SEQ ID NO：13)
Artificial sequence template primer 1 (511 site T:C sudden change):
5′-TTGTTCGCCA TTCCGTTCGC ATACTACATC TTGGACCATG AATTGGCTCG-3′(SEQ IDNO：14)
Artificial sequence template primer 2 (513 site A:T sudden change):
5′-TTGTTCGCCA TTCCGTTCGC ATACTACATT CGGGTTCTCG TCCATGAATA-3′(SEQ ID NO：15)
Artificial sequence template primer 3 (516 site A:T sudden change):
5′-TTGTTCGCCA TTCCGTTCGC ATACTACTAT TGACAGCGGG TTGTTCTGGA-3′(SEQ ID NO：16)
Artificial sequence template primer 4 (516 site G:T sudden change):
5′-TTGTTCGCCA TTCCGTTCGC ATACTACATT GACAGCGGGT TGTTCTGGTA-3′(SEQ ID NO：17)
Artificial sequence template primer 5 (being the contrast primer of artificial sequence template primer 1):
5′-TTGTTCGCCA TTCCGTTCGC ATACTACATC TTGGACCATG AATTGGCTCA-3′(SEQ ID NO：18)
Artificial sequence template primer 6 (being the contrast primer of artificial sequence template primer 2):
5′-TTGTTCGCCA TTCCGTTCGC ATACTACAGG GTTGTTCTCG TCCATGAATT-3′(SEQ ID NO：19)
Artificial sequence template primer 7 (being the contrast primer of artificial sequence template primer 3):
5′-TTGTTCGCCA TTCCGTTCGC ATACTACTAT TGACAGCGGG TTGTTCTGGT-3′(SEQ ID NO：20)
Artificial sequence template primer 8 (being the contrast primer of artificial sequence template primer 4):
5′-TTGTTCGCCA TTCCGTTCGC ATACTACATT GACAGCGGGT TGTTCTGGTC-3′(SEQ ID NO：21)
Artificial sequence template primer 9 (526 site A:C sudden change):
5′-TTGTTCGCCA TTCCGTTCGC ATACTACTCA TTGCTGTCGA GGTTGACCCC-3′(SEQ ID NO：22)
Artificial sequence template primer 10 (526 site C:T sudden change):
5′-TTGTTCGCCA TTCCGTTCGC ATACTACTCA TTCGCTGTCG TGGTTGACCT-3′(SEQ ID NO：23)
Artificial sequence template primer 11 (526 site C:A sudden change):
5′-TTGTTCGCCA TTCCGTTCGC ATACTACTCA TTCGCTGTCG AGGTTGACCA-3′(SEQ ID NO：24)
Artificial sequence template primer 12 (526 site C:G sudden change):
5′-TTGTTCGCCA TTCCGTTCGC ATACTACTCA TTCGCTGTCG AGGTTGACCG-3′(SEQ ID NO：25)
Artificial sequence template primer 13 (526 site A:G sudden change):
5′-TTGTTCGCCA TTCCGTTCGC ATACTACTCA TTGCTGTCGA GGTTGACCCG-3′(SEQ ID NO：26)
Artificial sequence template primer 14 (526 site C:G sudden change):
5′-TTGTTCGCCA TTCCGTTCGC ATACTACTCT TGCTGTCGAG GTTGACCCAG-3′(SEQ ID NO：27)
Artificial sequence template primer 15 (5311 site C:T sudden change):
5′-TTGTTCGCCA TTCCGTTCGC ATACTACTCA TTCCTCAAGC GCCGACTGTT-3′(SEQ ID NO：28)
Artificial sequence template primer 16 (531 site C:G sudden change):
5′-TTGTTCGCCA TTCCGTTCGC ATACTACTCA TTCCTCAAGC GCCGACTGTG-3′(SEQ ID NO：29)
Artificial sequence template primer 17 (the contrast primer of artificial sequence template primer 9,13):
5′-TTGTTCGCCA TTCCGTTCGC ATACTTACTC ATTGCTGTCG AGGTTGACCC-3′(SEQ ID NO：30)
Artificial sequence template primer 18 (artificial sequence template primer 10,11,12 contrast primer):
5′-TTGTTCGCCA TTCCGTTCGC ATACTACTCA TTGCTGTCGA GGTTGACCCA-3′(SEQ ID NO：31)
Artificial sequence template primer 19 (the contrast primer of artificial sequence template primer 14):
5′-TTGTTCGCCA TTCCGTTCGC ATACTCTCAT TGCTGTCGAG GTTGACCCAC-3′(SEQ ID NO：32)
Artificial sequence template primer 20 (the contrast primer of artificial sequence template primer 15,16):
5′-TTGTTCGCCA TTCCGTTCGC ATACTACTCA TTCCTCAAGC GCCGACTGTC-3′(SEQ ID NO：33)
The Taqman probe sequence that uses:
5′-TTGTTCGCCA TTCCGTTCGC-3′(SEQ ID NO：34)
Use therein reporter group is 6-Fluoresceincarboxylic acid (6-FAM) (being positioned at 5 ' end), and the quencher group is 6-carboxyl-tetramethyl-rhodamine (TAMRA) (being positioned at 3 ' end).
2, PCR scheme
The PCR scheme: 94 ℃, 5 minutes; 94 ℃ of 20 seconds and 61 ℃ 40 seconds, totally 40 circulations.Use therein sample is wild-type and each the mutant sequence that is cloned on the pUC plasmid, above-mentioned artificial sequence template primer collection is added 96 hole pcr amplification substrates, and the use transparent cover, expand at ABI GeneAmp 5700 fluorogenes and to increase on the ‰ ^ instrument and detect in real time.Its excitation light source is a halogen lamp, and wavelength is 488nm.Increase at the ABI5700 gene-amplificative instrament with above-mentioned primer and corresponding Taqman probe.
3, discrimination model
Judge whether to produce sudden change with different samples in the cycle number (Ct) that ABI5700 fluorescent PCR instrument detects fluorescence.The result is as shown in the table, and Ctwc refers to that wild-type sample (not sudden change) adds the cycle number that reaction obtains in the contrast artificial sequence template primer collection (not mutant primer); Ctwe refers to that wild-type sample (not sudden change) adds the middle cycle number that obtains of reacting of artificial sequence template primer collection (containing mutant primer) of sudden change; The Ctmc phalangeal process product that change add the cycle number that reaction obtains in the contrast artificial sequence template primer collections (not mutant primer): the Ctme phalangeal process product that change add in the artificial sequence template primer collection (containing mutant primer) of sudden change and react the cycle number that obtains.
| Contrast primer (c) | Mutant primer (e) | |
| Wild plasmid (w) | Ctwc | Ctwe |
| Mutant plasmid (m) | Ctmc | Ctme |
Ctwc＞Ctwe, Ctme＞Ctmc is a sufficient condition for what judge positive findings; Ctwc＞Ctmc, Ctwe＜Ctme is for judging the prerequisite of positive findings.
Satisfy or sufficient condition and prerequisite all satisfy as above-mentioned sufficient condition, then be judged to the positive (sudden change of detection is correct); As not satisfying sufficient condition, be judged to feminine gender (sudden change of detection does not take place); As satisfy sufficient condition, and prerequisite does not satisfy, and need be optimized primer.
4, result
With wild-type template template in contrast, mutant nucleotide sequence is an experiment pattern.Add the template of equivalent and the corresponding primer of equivalent.On the ABI5700 quantitative real time PCR Instrument, increase and fluorescence detect in real time.The reacting hole that adds different samples fluorescent signal (Ct＜40) occurs or fluorescent signal (Ct＞40) do not occur in difference circulation (Ct).According to above-mentioned discrimination model, detect each catastrophe point, and determine the type of sudden change.
Whether experimental result conforms to expection, promptly can discern different detected sudden changes and take place under same amplification condition, where occurs in, and what kind of sudden change has taken place.This platform technology illustrates the feasibility of homogeneous genetic matrix technology in the successful realization of existing 96 hole PCR substrates at present, promptly can be used for the detection of extensive, high-throughout single nucleotide polymorphism (SNPs), transgenation.
Embodiment 8
The two sudden change of tubercule bacillus rpoB SNPs detection technique platform is set up
Some special sudden change is taking place, and when suddenling change as C:T, mutant primer is little with the efficiency change of contrast wild-type primer amplification, and less than 10-2, whether the sudden change that is not easily distinguishable during detection (need do a large amount of optimizations) takes place.To make PCR be easy to distinguish this class sudden change in order reaching, when the design mutant primer, except that 3 ' terminal base is undergone mutation, can to carry out artificial mutation at its second of 3 ' end or the 3rd bit base.Results of mutation makes this primer when the amplification mutant nucleotide sequence, between primer and template, and its 3 ' terminal bases complementation, but second or tertiary base mispairing, primer still can extend forward and the amplified target sequence; But this primer is when the amplification wild-type sequence, and except that primer 3 ' end and template sequence did not match, its adjacent second or the 3rd bit base did not match yet, and it can't be increased to the wildtype target sequence.Otherwise, when the wild-type design of primers, add second or tertiary artificial mutation, can make this primer wild-type template that only increases, and the sample sequence of amplification sudden change hardly.
In this example, we select the C:T sudden change conduct detection target position in 531 sites of TB rpoB gene.
Primer (containing public primer):
Artificial sequence template primer 1:(contains 531 site C:T sudden change, and 3 ' terminal second T that suddenly change is A)
5′-ACAGACCAGA GACCCAGAGA TTGTTCGCCA TTCCGTTCGC ATACTACTCA TTCCTCAAGCGCCGACTGAT-3′(SEQ ID NO：35)
Artificial sequence template primer 2:(wild-type primer, artificial mutation 3 ' second T of end is A)
5′-ACAGACCAGA GACCCAGAGA TTGTTCGCCA TTCCGTTCGC ATACTACTCA TTCCTCAAGCGCCGACTGAC-3′(SEQ ID NO：36)
Public primer:
5′-ACAGACCAGA GACCCAGAG-3′(SEQ ID NO：37)
Conventional primer:
5′-GGCACGCTCA AGTGACAGAC-3′(SEQ ID NO：13)
The Taqman probe sequence that uses:
5′-TTGTTCGCCA TTCCGTTCGC-3′(SEQ ID NO：34)
Use therein reporter group is 6-Fluoresceincarboxylic acid (6-FAM) (being positioned at 5 ' end), and the quencher group is 6-carboxyl-tetramethyl-rhodamine (TAMRA) (being positioned at 3 ' end).
1, the PCR scheme is with embodiment 7.
2, discrimination model is with embodiment 7.
3, result
After two sudden change designs, corresponding to the sudden change template, the amplification efficiency of artificial sequence template primer 1 is apparently higher than the amplification efficiency of artificial sequence template primer 2 (Ct=40).For the wild-type template, the amplification efficiency of artificial sequence template primer 1 (Ct=40) then is starkly lower than the amplification efficiency of artificial sequence template primer 2.Can directly distinguish sudden change and whether exist, and which kind of sudden change takes place.
Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉Cao defends
＜120〉homogeneous genetic matrix
<130>014339
<160>37
<170>PatentIn version 3.0
<210>1
<211>50
<212>DNA
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aaggaacagg cggcgacgaa tcaacgacag aacgcaaccc aacgctactc 50
<210>2
<211>16
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<400>2
gtgcccccgc aagact 16
<210>3
<211>20
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<400>3
tcgtcgccgc ctgttcctta 20
<210>4
<211>50
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aaggaacagg cggcgacgaa tcaacgacag aaccagcgat agccaggaca 50
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cctccaatca ctcaccaacc 20
<210>6
<211>70
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tcatccacat cccacctcat caggaacagg cggcgacgaa tcaacgacag aacgcaaccc 60
aacgctactc 70
<210>7
<211>20
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tcatccacat cccacctcat 20
<210>8
<211>22
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tgagtgtcgt acagcctcca gg 22
<210>9
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tcatccacat cccacctcat caggaacagg cggcgacgaa tcatccagtc tatgtttccc 60
tcttgttgct 70
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agtttccgtc cgaaggttt 19
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aaggaacagg cggcgacgaa tcaacgacag aacgcaaccc aacgctactc 50
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caatcaagga gttcttcggc a 21
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ggcacgctca agtgacagac 20
<210>14
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ttgttcgcca ttccgttcgc atactacatc ttggaccatg aattggctcg 50
<210>15
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ttgttcgcca ttccgttcgc atactacatt cgggttctcg tccatgaata 50
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ttgttcgcca ttccgttcgc atactactat tgacagcggg ttgttctgga 50
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ttgttcgcca ttccgttcgc atactacatt gacagcgggt tgttctggta 50
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ttgttcgcca ttccgttcgc atactacatc ttggaccatg aattggctca 50
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ttgttcgcca ttccgttcgc atactacagg gttgttctcg tccatgaatt 50
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ttgttcgcca ttccgttcgc atactactat tgacagcggg ttgttctggt 50
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ttgttcgcca ttccgttcgc atactacatt gacagcgggt tgttctggtc 50
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ttgttcgcca ttccgttcgc atactactca ttgctgtcga ggttgacccc 50
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ttgttcgcca ttccgttcgc atactactca ttcgctgtcg tggttgacct 50
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ttgttcgcca ttccgttcgc atactactca ttcgctgtcg aggttgacca 50
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ttgttcgcca ttccgttcgc atactactca ttcgctgtcg aggttgaccg 50
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<212>DNA
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ttgttcgcca ttccgttcgc atactactca ttgctgtcga ggttgacccg 50
<210>27
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<212>DNA
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<400>27
ttgttcgcca ttccgttcgc atactactct tgctgtcgag gttgacccag 50
<210>28
<211>50
<212>DNA
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<400>28
ttgttcgcca ttccgttcgc atactactca ttcctcaagc gccgactgtt 50
<210>29
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ttgttcgcca ttccgttcgc atactactca ttcctcaagc gccgactgtg 50
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ttgttcgcca ttccgttcgc atacttactc attgctgtcg aggttgaccc 50
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ttgttcgcca ttccgttcgc atactactca ttgctgtcga ggttgaccca 50
<210>32
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ttgttcgcca ttccgttcgc atactctcat tgctgtcgag gttgacccac 50
<210>33
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ttgttcgcca ttccgttcgc atactactca ttcctcaagc gccgactgtc 50
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ttgttcgcca ttccgttcgc 20
<210>35
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acagaccaga gacccagaga ttgttcgcca ttccgttcgc atactactca ttcctcaagc 60
gccgactgat 70
<210>36
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acagaccaga gacccagaga ttgttcgcca ttccgttcgc atactactca ttcctcaagc 60
gccgactgac 70
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<212>DNA
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acagaccaga gacccagag 19
Claims (10)
1. homogeneous genetic matrix is characterized in that it comprises:
One substrate has 4-100 on the described substrate, 000 reacting hole;
In at least one described reacting hole, has artificial sequence template primer collection, described primer collection comprises: it is right that specificity is incorporated into detected nucleotide sequence and causes the Oligonucleolide primers of purpose nucleic acid amplification reaction, wherein at least one primer is an artificial sequence template primer, and this artificial sequence template primer comprises:
(a) specific combination district, this specific combination district is positioned at artificial sequence template primer 3 ' end, is used for combining with detected nucleotide sequence specificity;
(b) public domain, this public domain is positioned at 5 of artificial sequence template primer ' hold and has reporter molecules land and public guiding region, but the reporter molecules specificity is incorporated into the complementary strand of this report molecule land or this report molecule land, and the state difference of described reporter molecules under following two kinds of situations, thereby produce detectable signal: (i) be incorporated into the complementary strand of reporter molecules land or this report molecule land and (ii) reporter molecules be cut off or be substituted.
2. homogeneous genetic matrix as claimed in claim 1 is characterized in that, also is provided with the check plot on the described substrate, and there is at least one reacting hole described check plot, is used to place contrast agents.
3. homogeneous genetic matrix as claimed in claim 1 is characterized in that, described substrate is 96 orifice plates or fibre bundle, and described fibre bundle is made up of the respond optical fiber in pond of end.
4. homogeneous genetic matrix as claimed in claim 1 is characterized in that, also comprises a closure member, is used for the reacting hole of closed substrate.
5. homogeneous genetic matrix as claimed in claim 4 is characterized in that, described closure member is film, transparent cover, paraffin.
6. homogeneous genetic matrix as claimed in claim 1 is characterized in that, described reporter molecules comprises: FRET (fluorescence resonance energy transfer) type signal probe and contain the oligonucleotide chain of rare earth element.
7. homogeneous genetic matrix as claimed in claim 6, it is characterized in that, described specific combination section length is 6-35bp, the length of described public domain is 8-100bp, and described FRET (fluorescence resonance energy transfer) type signal probe is selected from down group: Taqman probe, the two probes of FRET, molecular beacon and PNA signal probe, and between specific combination district and reporter molecules land, there is transcribed spacer, and/or between public guiding region and reporter molecules land, having transcribed spacer, described transcribed spacer is 1-20bp.
8. homogeneous genetic matrix as claimed in claim 1, it is characterized in that, in described artificial sequence template primer, contain and be selected from the down Nucleotide of group: isoG, isoC, 2 '-O-methyl-G, 2 '-O-methyl-C, antigen molecule or biotin labeled Nucleotide and combination thereof.
9. homogeneous genetic matrix as claimed in claim 1 is characterized in that, described artificial sequence template primer collection also comprises the amplification template primer that is used to increase detected nucleic acid-templated quantity.
10. nucleic acid detection method is characterized in that it comprises step:
(1) sample is added to a substrate, have 4-100 on the described substrate, 000 reacting hole; In at least one described reacting hole, has artificial sequence template primer collection, described primer collection comprises: it is right that specificity is incorporated into detected nucleotide sequence and causes the Oligonucleolide primers of purpose nucleic acid amplification reaction, wherein at least one primer is an artificial sequence template primer, and this artificial sequence template primer comprises:
(a) specific combination district, this specific combination district is positioned at artificial sequence template primer 3 ' end, is used for combining with detected nucleotide sequence specificity;
(b) public domain, this public domain is positioned at 5 of artificial sequence template primer ' hold and has reporter molecules land and public guiding region, but the reporter molecules specificity is incorporated into the complementary strand of this report molecule land or this report molecule land, and the state difference of described reporter molecules under following two kinds of situations, thereby produce detectable signal: the complementary strand that (i) is incorporated into reporter molecules land or this report molecule land, (ii) reporter molecules is cut off or is substituted
(2) under the specificity condition, carry out nucleic acid amplification reaction,
(3) detectable signal that produced of examining report molecule.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011265787A CN1186457C (en) | 2001-08-29 | 2001-08-29 | Homogeneous genetic matrix |
| PCT/CN2002/000591 WO2003019189A1 (en) | 2001-08-29 | 2002-08-26 | Homogeneous phase gene microarray |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011265787A CN1186457C (en) | 2001-08-29 | 2001-08-29 | Homogeneous genetic matrix |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1407113A CN1407113A (en) | 2003-04-02 |
| CN1186457C true CN1186457C (en) | 2005-01-26 |
Family
ID=4666590
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB011265787A Expired - Lifetime CN1186457C (en) | 2001-08-29 | 2001-08-29 | Homogeneous genetic matrix |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN1186457C (en) |
| WO (1) | WO2003019189A1 (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4541731B2 (en) * | 2004-03-12 | 2010-09-08 | キヤノン株式会社 | Nucleic acid detection method |
| EP2128891B1 (en) | 2007-02-28 | 2015-09-02 | Shin-Etsu Chemical Co., Ltd. | Process for producing laminated substrate |
| KR20130110170A (en) | 2010-09-22 | 2013-10-08 | 앨리오스 바이오파마 인크. | Substituted nucleotide analogs |
| US8980865B2 (en) | 2011-12-22 | 2015-03-17 | Alios Biopharma, Inc. | Substituted nucleotide analogs |
| CN104321333A (en) | 2012-03-21 | 2015-01-28 | 沃泰克斯药物股份有限公司 | Solid forms of a thiophosphoramidate nucleotide prodrug |
| NZ630805A (en) | 2012-03-22 | 2016-01-29 | Alios Biopharma Inc | Pharmaceutical combinations comprising a thionucleotide analog |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1117282C (en) * | 1999-09-03 | 2003-08-06 | 何农跃 | PCR microarray probe circulating detection type biological chip |
| US6500620B2 (en) * | 1999-12-29 | 2002-12-31 | Mergen Ltd. | Methods for amplifying and detecting multiple polynucleotides on a solid phase support |
-
2001
- 2001-08-29 CN CNB011265787A patent/CN1186457C/en not_active Expired - Lifetime
-
2002
- 2002-08-26 WO PCT/CN2002/000591 patent/WO2003019189A1/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| WO2003019189A1 (en) | 2003-03-06 |
| CN1407113A (en) | 2003-04-02 |
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