CN101519698A - Method for quantitatively measuring nucleic acid with sequence tags - Google Patents
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- CN101519698A CN101519698A CN 200910126690 CN200910126690A CN101519698A CN 101519698 A CN101519698 A CN 101519698A CN 200910126690 CN200910126690 CN 200910126690 CN 200910126690 A CN200910126690 A CN 200910126690A CN 101519698 A CN101519698 A CN 101519698A
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Abstract
The invention discloses a method for quantitatively measuring nucleic acid with sequence tags, belonging to the filed of molecular genetics. The method comprises the steps of carrying out coupled reaction to tag nucleic acid to be measured, carrying out amplified reaction to increase the amount of production from the coupled reaction, and carrying out sequencing reaction to identify and quantify sequence tags on products of the coupled reaction. The method is characterized in that a single tube is internally provided with one pair or a plurality of pairs of connection probes, each pair of connection probes forms a specific complementary sequence with the nucleic acid to be measured, and at least one pair of connection probes is provided with specific sequence tags. The invention can be used for easily, conveniently and quantitatively measuring a plurality of nucleic acid without adopting the fluorescent dye tagging method but the base sequence tagging method with low cost and high flux so as to measure the gene sequence difference and the relative expression quantity of genes, thereby the invention can be clinically popularized and used widely.
Description
Technical field
The invention belongs to the molecular genetic field, relate to a kind of method of utilizing the sequence label quantitatively measuring nucleic acid.
Background technology
The nucleic acid quantification analysis is exactly the relative content of quantitative assay particular sequence nucleic acid, and the essence of gene order difference and gene expression amount variance analysis is exactly the quantitatively measuring nucleic acid template.
The difference of gene order comprises that mainly single nucleotide polymorphism (SNP), sequence repeat (copy number variation, CNV) and sequence deletion, wherein single nucleotide polymorphism be meant in the genomic dna change on a certain specific nucleotide position, variation such as transversion, insertion or disappearance.Gene order repeats and disappearance refers to certain exon sequence of the partial sequence of a fragment gene, a kind of gene, the full sequence of certain gene even the repetition or the disappearance of a whole group chromosome sequence.Gene expression amount difference detect be meant more same gene transcript mRNA under different cells, different organs tissue, different physiological status distribution and the difference of amount, be the important channel of carrying out the gene function parsing.Can according to measurement result infer unknown gene function, separate Benq because of between and protein between interaction.
Improve analysis throughput (i.e. the number of the sample number that once can measure or target to be measured) and can lower the detection cost and improve detection efficiency, and the multiple detectivity of raising method is to improve the important means of analysis throughput.Usually the polynary labeling technique by biomolecules realizes that multiple parallel detects.Biomolecules labeling technique commonly used is a fluorescent mark.Adopt the fluorescence dye of different colours to be subjected to the restriction of fluorescent substance kind and fluoroscopic examination wavelength region, can carry out mark with four kinds of fluorescent probes simultaneously at the most at present with a plurality of target molecules to be measured of tense marker
(1), and usually only with two kinds of a confidential reference items of fluorescence dyes difference mark and sample to be tested.In order to solve the deficiency of high throughput analysis aspect, developed coding techniques.A kind of is the chip dot matrix coding techniques of using always
(2), the probe that is about to encode is fixed on chip surface by microarray, and then carries out chip scanning and detect; Second method is based on the coding techniques of mixing colouring agent spectroscopy feature
(3,4), promptly elder generation prepares serial coding microball carrier with the fluorescence dye of two kinds of colors according to different mixed, and at the fixing different probe of microsphere surface, treats the micrometer ball according to the detected fluorescence intensity ratio in different wave length place then and decode; The third method is based on the coding method of quantum dot
(5,6), the quantum dot that is about to different-grain diameter is embedded in the polymkeric substance by different ratios, obtains the microballoon of different coding, and at its finishing dna probe
(7,8)Adopt the different organic molecule of the molecular weight different nucleic acid molecule of also can encoding, but need the mass-spectrometric technique decoding, not only loaded down with trivial details but also time-consuming, the effort of this method, need discharge marker by chemical reaction during detection could detect.
Lack at present a kind of simple, convenient, flux is high, cost is lower and can be used to measure the method that gene order difference can be used to measure the gene relative expression quantity again.
Reference:
1.Vet，J.A.，Van?der?Rijt，B.J.and?Blom，H.J.(2002)Molecular?beacons：colorful?analysis?ofnucleic?acids.Expert?review?of?molecular?diagnostics，2，77-86.
2.Duggan，D.J.，Bittner，M.，Chen，Y.，Meltzer，P.and?Trent，J.M.(1999)Expression?profiling?usingcDNA?microarrays.Nat?Genet，21，10-14.
3.Schmitt，M.，Bravo，I.G.，Snijders，P.J.，Gissmann，L.，Pawlita，M.and?Waterboer，T.(2006)Bead-based?multiplex?genotyping?of?human?papillomaviruses.J?Clin?Microbiol，44，504-512.
4.Yang，L，Tran，D.K.and?Wang，X.(2001)BADGE，Beads?Array?for?the?Detection?of?GeneExpression，a?high-throughput?diagnostic?bioassay.Genome?Res，11，1888-1898.
5.Cao，Y.C.，Huang，Z.L，Liu，T.C.，Wang，H.Q.，Zhu，X.X.，Wang，Z.，Zhao，Y.D.，Liu，M.X.andLuo，Q.M.(2006)Preparation?of?silica?encapsulated?quantum?dot?encoded?beads?for?multiplex?assay?and?itsproperties.Anal?Biochem，351，193-200.
6.Gao，X.and?Nie，S.(2005)Quantum?dot-encoded?beads.Methods?Mol?Biol，303，61-71.
7.Vaidya，S.V.，Gilchrist，M.L.，Maldarelli，C.and?Couzis，A.(2007)Spectral?bar?coding?ofpolystyrene?microbeads?using?multicolored?quantum?dots.Anal?Chem，79，8520-8530.
8.Han，M.，Gao，X.，Su，J.Z.and?Nie，S.(2001)Quantum-dot-tagged?microbeads?for?multiplexedoptical?coding?of?biomolecules.Nat?Biotechnol，19，631-635.
Summary of the invention
The purpose of this invention is to provide a kind of simple, convenient, flux is high, cost is lower, do not adopt the method for fluorochrome label and adopt the sequence label technology to set up and a kind ofly can detect the method that a plurality of gene order differences can be measured the gene relative expression quantity simultaneously in single tube.
Ultimate principle of the present invention is at first to adopt probe and the purpose target hybridization that contains sequence label, ligation takes place under the effect of enzyme form the connection product that contains sequence label, adopt a pair of general primer to carry out pcr amplification reaction, measure sequence label with burnt sequencing technologies at last, can carry out quantitative analysis to gene order difference and gene expression amount difference according to the order-checking measurement result.
The objective of the invention is to realize by following technical measures:
A kind of method of utilizing base sequence label technique quantitatively measuring nucleic acid, comprise the following steps: that ligation mark determined nucleic acid, amplified reaction increase the sequence label on ligation product amount, sequencing reaction identification and the quantitative ligation product, it is characterized in that containing in the single tube one or more pairs of linking probes, there are the specificity complementary sequence in every pair of linking probe and a kind of determined nucleic acid, and this contains specific sequence label at least one probe in the linking probe.
Described method, wherein sequence label is meant the dna fragmentation with Different Alkali basic sequence.Be marked on the probe in order to discern different determined nucleic acid targets.The sequence label length that is used for mark is identical, and the Tm value is identical, only the base difference that puts in order.
Described method, wherein ligation is meant under the effect of ligase enzyme and the reaction that takes place with a pair of adjacent linking probe of a kind of sequence generation complementary of determined nucleic acid.
Described method, wherein a pair of adjacent linking probe are meant two probes and with behind a kind of determined nucleic acid molecular hybridization, wherein 5 ' end of 3 ' of linking probe end and another linking probe is closely adjacent.
Described method, wherein ligation is meant in single tube many to the simultaneous ligation of adjacent linking probe.
Described method, wherein linking probe one end contain one section with determined nucleic acid template complementary sequence, the other end contain one section with determined nucleic acid template complementary sequence not.
Described method, wherein with the determined nucleic acid template not the complementary sequence in the probe that does not contain sequence label, comprise one section PCR primer specificity sequence, can also comprise one section restriction endonuclease specificity site sequence; With the determined nucleic acid template not the complementary sequence in containing the probe of sequence label, comprise one section PCR primer specificity sequence and sequence label, can also comprise one section restriction endonuclease specificity site sequence.Described restriction endonuclease is meant IIS type restriction endonuclease, and the PCR primer is meant the general primer of the connection product that is used to increase corresponding with multiple nucleic acid.
Described method, wherein sequencing reaction is meant the sequencing technologies (being burnt sequencing reaction) of measuring the pyrophosphate salt of primer extension generation based on bioluminescence reaction.
Described method, wherein sequencing reaction is meant the sequencing reaction that adopts dNTP to carry out, or the sequencing reaction that adopts ddNTP to carry out.
The concentration and probe concentration of ligation is 0.1-5nmol/L.
The preferred 2-40 kind of multiple nucleic acid nucleic acid.
Amplified reaction is meant and adds the cyclic amplification reaction that restriction endonuclease carries out the constant temperature endonuclease reaction and carry out sex change, annealing, extension again after high temperature deactivation.
Beneficial effect of the present invention:
The present invention adopts the base sequence labelling method to replace fluorescent marker method, does not need laser and fluorescence dye, does not also use electrophoresis.A plurality of different targets to be measured are labeled different sequence labels simultaneously in single tube, and it is identical to be used for the sequence label length of mark, and the Tm value is identical, only the base difference that puts in order.With compare with sequence length difference labelling method, the probe length unanimity in this law, amplification efficiency is close, the probe price is also low.
Adopt in single tube, increase the simultaneously connection product of Different Alkali basic sequence on a plurality of being labeled of a pair of general primer, compare with traditional multiple PCR technique, do not have the phase mutual interference between primer, and can adopt the linking probe (fmol level) of lower concentration to reduce the interference of multiprobe PCR.
Target detection by quantitative method to be measured of the present invention has general applicability, promptly can detect the difference (SNP, CNV etc.) of range gene sequence by primary first-order equation simultaneously, can detect the relative expression quantity of a plurality of genes again.
Gene order difference that the present invention can detect in single tube simultaneously is a plurality of (be unlimited in theory, in the practice preferred 20-40) (contain SNP, sequence repeats and disappearance) and a plurality of gene (be unlimited in theory, in the practice preferably 10-20 kind).
Method provided by the invention is simple, convenient, flux is high, cost is lower and can be used to measure gene order difference can be used to again measure the gene relative expression quantity, helps clinically extensively promoting the use of.
Description of drawings
Fig. 1: the synoptic diagram of measuring two kinds of determined nucleic acid targets with the sequence label method.
Fig. 2: the sequence label decoding principle of a plurality of targets to be measured.
Fig. 3: measure single base polymorphisms with the sequence label method.
Fig. 4: 6 genes (Beta-actin, C-myc, CD44v6, H-ras, COX-2, the measurement results of relative expression quantity RAB5A) in the Colorectal Carcinoma.
Embodiment
The invention will be further elaborated by the following examples.
Embodiment 1: the method for two kinds of determined nucleic acids of quantitative assay
As shown in Figure 1, determination step of the present invention is as follows:
(1) adopts the base sequence labelling method, make different targets be labeled different sequences
At first, design a pair of probe 3,4 (corresponding to target 1) and a pair of probe 5,6 (corresponding to target 2) respectively according to the gene order 1 and 2 of template A to be measured and B.Every probe is made up of two portions, a part and template complementation, and another part contains and template complementary sequence not.In every pair of probe, there is 5 ' end of a probe closely to link to each other, when template exists, under the effect of ligase enzyme 7, ligation takes place with another 3 ' end; X in the probe 3 and 5 for not with the template complementary sequence, identical with the primer of follow-up PCR reaction; In the probe 4 and 6 with template not the complementary sequence form by three parts, sequence Y (as shown in the figure), sequence B and IIS restriction enzyme site sequence, the wherein primer complementation of sequence Y and follow-up PCR reaction, sequence B is a sequence label, and different targets contains different sequence B, in order to keep the efficient of pcr amplification reaction, sequence B can be designed to base sequence respectively and arrange difference, but the kind of base is identical with number.Sequence B is relevant with the character of template to be measured, uses sequence B 1 and B2 mark target A and target B respectively.IIS restriction enzyme site sequence is used to eliminate the pollution of pcr template.(design of every pair of probe is carried out with reference to this method, and there are the specificity complementary sequence in every pair of probe and a kind of determined nucleic acid, and this contains specific sequence label at least one probe in the probe.When having a plurality of sequence label, each sequence label is different).After ligation is finished, carry out pcr amplification reaction with a pair of universal primer 8 and 9 (5 ' end of primer 8 is modified with biotin, is used to prepare the single stranded DNA template).The sequence Tm value of PCR product also can suitably be adjusted as the case may be.
(2) based on the real-time noclilucence sequencing technologies of pyrophosphate salt amplified production is carried out the base sequence decoding
By aforesaid method in single tube with a pair of primer a plurality of gene target that increase simultaneously, and every kind of target all is marked with different base sequence B, it is crucial how these amplified productions being decoded.The present invention adopts the real-time bioluminescence assay technology of pyrophosphate salt that amplified production is carried out the base sequence decoding, promptly the microballoon of modifying with streptavidin earlier prepares single stranded DNA 11 and 12, adding sequencing primer 13 again anneals with it, primer 13 can be corresponding with the partial sequence in the sequence B, promptly adopts the sequence of primer 13 and the extension sequence of primer 13 jointly the sequence label of target to be measured to be decoded.Add different dNTP during order-checking respectively successively and carry out burnt sequencing reaction, obtain among the figure with respect to the collection of illustrative plates of target A and B, its peak height is the relative content of two targets to be measured.
Can realize the mensuration of gene order difference and gene expression amount by designing different probes.Concrete grammar is as follows:
SNP (single base polymorphisms) measures: SNP has two kinds of allelotrope (coming from paternal and maternal respectively) usually, every type corresponding to a kind of template, promptly two kinds of templates (target to be measured) have only a base sequence difference, these two kinds of templates can be corresponded respectively to Target A and Target B among Fig. 1.If the sequence B in Fig. 1 middle probe is used for the type of marker allele, if template to be measured is a homozygote, then have only a peak to occur, if during heterozygote, two peaks that intensity equates appear.Also can be with one section sequence label that is used for different positions SNP site on the marker gene group of design in the sequence B, to realize the same tense marker in a plurality of SNP site.
CNV (copy number variation) measures: when measuring the gene copy number variation, must be the actual copy number that basis of reference (promptly not having the variation of copy number) is measured unknown template with one section sequence.Can be with the Target A among Fig. 1 as reference, Target B is a target to be measured.If the sequence B on Fig. 1 middle probe 1 is used for targets to be measured different on the marker gene group with B2, the relative height at two peaks is the changing conditions of two testing gene fragment copy numbers in the measurement result.
The genetic expression flow measurement: when measuring the expression amount of a plurality of genes in a certain tissue, must a kind of house-keeping gene be basis of reference, the expression amount of this genoid in various tissues be constant substantially, can regard constant basis as.During mensuration, can be with the Target A among Fig. 1 as reference (house-keeping gene), Target B is target (testing gene).If sequence B on Fig. 1 middle probe 1 and B2 are used for the marker gene kind, the relative height at two peaks is the relative expression quantity (being the template amount) of marker gene in the measurement result.
Embodiment 2: the method that the sequence label of a plurality of targets to be measured is decoded simultaneously
The method principle: when adopting dyes in different colors that different targets to be measured is carried out mark, be subjected to the restriction of dyestuff kind and wavelength region, can not be with the target of tense marker more than 4, the present invention can reach with the target of tense marker more than 10 by sequence label.As shown in Figure 2, be measured as example in the time of with 6 different targets, with 6 kinds of different targets of three base sequence marks, in the order-checking collection of illustrative plates, a kind of target is corresponding with a sequence peak.Can progressively extend owing to measure sequence, must use and isolate base, otherwise four kinds of different bases can only three kinds of targets of mark, as CA, GA and TA.We are used for mark (C and G) with two kinds in four kinds of bases, and other two kinds (A and T) is used for isolating, and measure when having realized a plurality of target like this.
In Fig. 2, use CAT, GAT, TCT, TGT, TAC, TAG be six kinds of targets of mark respectively, when adding dCTP, have only the sequence label corresponding with target 1 to go out peak (left side pentagram among Fig. 2); Same when adding dGTP, have only the sequence label corresponding to go out peak (left side cross star among Fig. 2) with target 2; Add dTTP (isolate and use) then, except that target 1 and 2, extension has all taken place in all the other four kinds of targets, can obviously find out the peak of T among Fig. 2; When add adding dCTP again, have only the sequence label corresponding to go out peak (pentagram in the middle of among Fig. 2) with target 3; Same when adding dGTP, have only the sequence label corresponding to go out peak (cross star in the middle of among Fig. 2) with target 4; Add dATP (isolate and use) then, except that target 3 and 4, extension has all taken place in all the other four kinds of targets, can obviously find out the peak of A among Fig. 2; When add adding dCTP again, have only the sequence label corresponding to go out peak (the right pentagram among Fig. 2) with target 5; Same when adding dGTP, have only the sequence label corresponding to go out peak (the right cross star among Fig. 2) with target 6.Like this, measure when once burnt order-checking has just realized a plurality of target.
By that analogy, the sequence of 6 bases of employing just can 12 kinds of different targets of mark.Can measure infinitely a plurality of in theory.
Embodiment 3: the mensuration of the multiple single base polymorphisms relevant with diabetes
1. experiment material
Reagent: genomic dna, by extracting in patient's blood sample; Ampligase (EPICENTRE); The Taq archaeal dna polymerase (TaKaRa Biotechnology Co., Ltd); DNTPs (Promega, Madison, USA); Primer LDR-P1:5 '-cat ctg ttc cct ccc tgt c-3 '; LDR-P2:5 '-biotin-ccc cac ttc ttg ttc tct cat-3 ' (Invitrogen Inc., Shanghai, PAGE level); Agarose (OXOID Ltd., Hampshire, England); Gel electrophoresis application of sample liquid and ethidium bromide (EB) are Sangong biotech firm product.Dynabeads M-280-streptavidin (Dynal AS, Oslo, Norway), 1 * B﹠amp; The W damping fluid (10mmol/L Tris-HCl, 1mmol/L EDTA, 1.0mol/L NaCl, pH7.5), 0.1mol/L NaOH, 1 * annealing buffer (10mmol/L Tris-HCl, 1mmol/L EDTA, pH8.0), 1mol/L HCl.
Instrument: clean bench (VD-650-U type, the safe and sound technology in the sky in Suzhou company limited); Whizzer (PMC-860 type, CAPSULEFUGE company); Pcr amplification instrument (PTC-225 type, MJ Research company); High-voltage power supply electrophoresis apparatus (Power PAC1000, BIO-RAD, the U.S.); Electrophoresis chamber (laboratory assembling); Gel electrophoresis imager (Chemilmager Tm type, AlpaHa Innoteach company, the U.S.).Burnt sequenator (laboratory assembling).
2. experimental procedure
Ligation: contain genomic dna template (four kinds of SNP sites relevant in the 10 μ l reaction systems with diabetes, it is respectively the C1431T site of PPAR gene, the C2821T site of PPAR gene, the Prol2Ala site of PPARG gene, the Arg1273Arg site of SUR1 gene, there are three typical genotype dna profilings in each site) (100～500ng), (probe is corresponding with these 12 determined nucleic acid templates respectively, and 4*3=12 is right altogether for probe mixture, every kind of 50fmol, the preparation method is referring to embodiment 1), Ampligase 1U, 20mM Tris-HCl (pH8.3), 25mM KCl, 10mM MgCl2,0.5mM NAD, 0.01% Triton X-100.Reaction conditions is: 98 ℃ of pre-sex change 5min; 95 ℃ of sex change 1min then, connect 4min at 60 ℃ of annealing, carry out 10 circulations.
Polymerase chain reaction: contain primer (200nM) in the 25 μ l reaction systems, Tris-HCl (pH8.3) (10mM), KCl (50mM), MgCl2 (1.5mM), dNTPs (0.2mM), rTaq enzyme (0.75U).Reaction conditions is: 95 ℃ of pre-sex change 5min; With 95 ℃ of 30s, 60 ℃ of 1min, carry out 35 circulations then.
Burnt order-checking: at first prepare strand, the first step is got the magnetic microsphere of 1/10 PCR volume, inhales with magnet and abandons supernatant, uses B﹠amp; W damping fluid washing 2 times is suspended in an amount of B﹠amp; In the W damping fluid; The PCR product is mixed with magnetic microsphere, put jolting 1h in 37 ℃; Supernatant is abandoned in suction, and water washing 2 times adds the NaOH mixing of the 0.1mol/L of 20 μ L, places 5min; Supernatant is abandoned in suction, again with using B﹠amp after the 0.1mol/L NaOH washing once; W damping fluid washing 2 times; The annealing primer that adds 1 * annealing buffer and the 10pmol of 10 μ L, 95 ℃ of preannealing 30s, 55 ℃ of annealing 3min then.In sequencing template, add sequencing reaction liquid, add dGTP, dCTP and dTTP successively and carry out sequencing reaction.
3. experimental result
Measured four kinds of SNP sites relevant with diabetes in a pipe simultaneously, Fig. 3 is the partial results of measuring three typical genomic dna samples, the i.e. measurement result in the C1431T site of PPAR gene.When the peak that has only the wild-type represented in the mensuration collection of illustrative plates occurred, the C1431T that shows sample to be tested PPAR gene was a wild-type; Except that the peak that the wild-type represented is arranged, represent the peak of mutant to occur in addition in measuring collection of illustrative plates, the C1431T site that shows sample to be tested PPAR gene is a heterozygote; When having only the peak of the mutant represented in measuring collection of illustrative plates, the C1431T that shows sample to be tested PPAR gene is a mutant.
Embodiment 4: the sequence label method is used for the mensuration of gene expression amount difference
Present embodiment adopt sequence label method of the present invention investigate in the Colorectal Carcinoma 5 genes (C-myc, CD44v6, H-ras, COX-2, expression RAB5A) is reference with house-keeping gene beta-actin.Sequence label in the linking probe as shown in Figure 2.
At first, 5 ' end Starch phosphorylase (the T4 polynucleotide kinase) phosphorylation (37 degree 30min) of the linking probe of 6 genes, 10min deactivation in the water-baths of 70 degree then will be measured.Add mixed probe (6 pairs) (every kind of 5fmol) and carry out ligation.Do pcr amplification with the template after connecting (10 times of template dilutions), with amplified production (preparation strand, carry out Jiao's order-checking, the result as shown in Figure 4, the relative detected result of each gene marks with square frame in Fig. 1, each square frame has two peaks, and second peak is the background signal of dNTP, and practical measurement result must deduct this background.The result shows that six kinds of tumor-related genes all have expression in large bowel cancer, and COX-2 expression of gene amount is the highest, but all expression of gene amounts are all low than house-keeping gene.
＜110〉Zhou Guohua
＜120〉a kind of method of utilizing the sequence label quantitatively measuring nucleic acid
<160>2
<210>1
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉upstream primer
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catctgttcc?ctccctgtc?19
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<211>21
<212>DNA
＜213〉artificial sequence
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＜223〉downstream primer
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ccccacttct?tgttctctca?t?21
Claims (10)
1. method of utilizing sequence label technology quantitatively measuring nucleic acid, comprise the following steps: that ligation mark determined nucleic acid, amplified reaction increase the sequence label on ligation product amount, sequencing reaction identification and the quantitative ligation product, it is characterized in that containing in the single tube one or more pairs of linking probes, there are the specificity complementary sequence in every pair of linking probe and a kind of determined nucleic acid, and this contains specific sequence label at least one probe in the linking probe.
2. method according to claim 1 is characterized in that sequence label is meant the dna fragmentation with Different Alkali basic sequence.
3. method according to claim 1 is characterized in that ligation is meant under the effect of ligase enzyme and the reaction that takes place with a pair of adjacent linking probe of a kind of sequence generation complementary of determined nucleic acid.
4. method according to claim 3 is characterized in that a pair of adjacent linking probe is meant two probes and with behind a kind of determined nucleic acid molecular hybridization, wherein 5 ' end of 3 ' of linking probe end and another linking probe is closely adjacent.
5. method according to claim 3, it is many to the simultaneous ligation of adjacent linking probe to it is characterized in that ligation is meant in single tube.
6. method according to claim 1, it is characterized in that linking probe one end contain one section with determined nucleic acid template complementary sequence, the other end contain one section with determined nucleic acid template complementary sequence not.
7. method according to claim 6, it is characterized in that with the determined nucleic acid template not the complementary sequence in the probe that does not contain sequence label, comprise one section PCR primer specificity sequence, can also comprise one section restriction endonuclease specificity site sequence; With the determined nucleic acid template not the complementary sequence in containing the probe of sequence label, comprise one section PCR primer specificity sequence and sequence label, can also comprise one section restriction endonuclease specificity site sequence.
8. method according to claim 7 is characterized in that restriction endonuclease is meant IIS type restriction endonuclease; The PCR primer is meant the general primer of the connection product that is used to increase corresponding with multiple nucleic acid.
9. method according to claim 1 is characterized in that sequencing reaction is meant the sequencing technologies of measuring the pyrophosphate salt of primer extension generation based on bioluminescence reaction.
10. method according to claim 9 is characterized in that sequencing reaction is meant the sequencing reaction that adopts dNTP to carry out, or the sequencing reaction that adopts ddNTP to carry out.
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| WO2002004680A2 (en) * | 2000-07-07 | 2002-01-17 | Visigen Biotechnologies, Inc. | Real-time sequence determination |
| CN1532291A (en) * | 2003-03-21 | 2004-09-29 | 中国人民解放军军事医学科学院放射医 | High efficiency quick detection method of ligonucleotide library small fragment sequence |
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