CN101519698B - Method for quantitatively measuring nucleic acid with sequence tags - Google Patents
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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 fluxso 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 quantitative analysis of nucleic acids 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 refer to change on a certain specific nucleotide position in genomic dna, the 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, repetition or the disappearance of an even whole group chromosome sequence.The gene expression amount Difference test refer to comparison same gene transcription product mRNA under different cells, different organs tissue, different physiological status distribution and the difference of amount, be to carry out the important channel that gene function is resolved.Can according to measurement result infer unknown gene function, separate Benq because of between reach interaction between protein.
Improve analysis throughput (sample number that namely once can measure or the number of target to be measured) and can lower testing cost and improve detection efficiency, and the Multiple detection ability 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 fluorescent mark.Adopt the fluorescence dye of different colours to be subjected to the restriction of fluorescent substance kind and fluoroscopic examination wavelength region with a plurality of target molecules to be measured of tense marker, can carry out mark with four kinds of fluorescent probes simultaneously at the most at present
(1), and usually only with two kinds of a internal references 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 commonly using
(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), namely first being mixed with serial coding microball carrier with the fluorescence dye of two kinds of colors according to different ratios, and at the fixing different probe of microsphere surface, the fluorescence intensity ratio that then detects according to the different wave length place is treated the micrometer ball and is decoded; 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 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, the method is loaded down with trivial details but also time-consuming, effort not only, and need to 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 for measuring gene order difference can be used for measuring the method for 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 simultaneously the method that a plurality of gene order differences can be measured the gene relative expression quantity 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 occurs 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 Pyrosequencing 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, comprising the following steps: that ligation mark determined nucleic acid, amplified reaction increase ligation product amount, sequencing reaction identification and be connected connects sequence label on reaction product, it is characterized in that containing in 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 linking probe.
Described method, wherein sequence label refers to have the DNA fragmentation of Different Alkali basic sequence.Be marked on probe in order to identify 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 refers to occur with the sequence of same determined nucleic acid the reaction of complementary a pair of adjacent linking probe generation under the effect of ligase enzyme.
Described method, after wherein a pair of adjacent linking probe referred to two probes and same determined nucleic acid molecular hybridization, wherein 5 ' end of 3 ' of linking probe end and another linking probe was closely adjacent.
Described method, wherein ligation refers in single tube many to the simultaneous ligation of adjacent linking probe.
Described method, wherein linking probe one end contains one section sequence with the complementation of determined nucleic acid template, and the other end contains one section sequence not complementary with the determined nucleic acid template.
Described method wherein comprises one section PCR primer specificity sequence with the not complementary sequence of determined nucleic acid template at the probe that does not contain sequence label, can also comprise one section restriction endonuclease specificity site sequence; The sequence not complementary with the determined nucleic acid template comprises one section PCR primer specificity sequence and sequence label at the probe that contains sequence label, can also comprise one section restriction endonuclease specificity site sequence.Described restriction endonuclease refers to IIS type restriction endonuclease, and the PCR primer refers to the general primer for the amplification connection product corresponding with multiple nucleic acids.
Described method, wherein sequencing reaction refers to measure based on bioluminescence reaction the sequencing technologies (being burnt sequencing reaction) of the pyrophosphate salt of primer extension generation.
Described method, wherein sequencing reaction refers to 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 acids nucleic acid.
Amplified reaction refers to the cyclic amplification reaction that adds restriction endonuclease to carry 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 in this law is consistent, amplification efficiency is close, the probe price is also low.
Adopt increase the simultaneously connection product of Different Alkali basic sequence on a plurality of being labeled of a pair of general primer in single tube, compare with traditional multiple PCR technique, there is no the phase mutual interference between primer, and can adopt the linking probe (fmol level) of lower concentration to reduce multiprobe to the interference of PCR.
Target detection by quantitative method to be measured of the present invention has general applicability, namely can detect simultaneously the difference (SNP, CNV etc.) of range gene sequence by primary first-order equation, can detect again the relative expression quantity of a plurality of genes.
It (is unlimited in theory that the present invention can detect a plurality of in single tube simultaneously, in practice, preferred 20-40 is individual) gene order difference (containing SNP, sequence repetition and disappearance) and a plurality of gene (being unlimited in theory, preferred 10-20 kind in practice).
Method provided by the invention is simple, convenient, flux is high, cost is lower and can be used for measuring gene order difference can be used for again measuring the gene relative expression quantity, is conducive to clinically extensively promote the use of.
Description of drawings
Fig. 1: the schematic 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: the measurement result of the relative expression quantity of 6 genes (Beta-actin, C-myc, CD44v6, H-ras, COX-2, RAB5A) in 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) adopt the base sequence labelling method, make different targets be labeled different sequences
At first according to the gene order 1 and 2 of template A to be measured and B, design respectively a pair of probe 3,4 (corresponding to target 1) and a pair of probe 5,6 (corresponding to target 2).Every probe is comprised of two portions, and a part is complementary with template, and another part contains the sequence not complementary with template.There is 5 ' end of a probe closely to be connected with another 3 ' end in every pair of probe, when having template to exist, under the effect of ligase enzyme 7, ligation occurs; X in probe 3 and 5 for not with the template complementary sequence, identical with the primer of follow-up PCR reaction; Formed by three parts with the not complementary sequence of template in probe 4 and 6, sequence Y (as shown in the figure), sequence B and IIS restriction enzyme site sequence, wherein the primer of sequence Y and follow-up PCR reaction is complementary, sequence B is 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 respectively base sequence and arrange difference, but the kind of base is identical with number.Sequence B is relevant to the character of template to be measured, uses respectively sequence B 1 and B2 mark target A and target B.IIS restriction enzyme site sequence is used for eliminating 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 probe.When having a plurality of sequence label, each sequence label is different).After ligation is completed, carry out pcr amplification reaction with a pair of universal primer 8 and 9 (5 ' end of primer 8 is modified with biotin, for the preparation of 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 pair of primers 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 to carry out the base sequence decoding to amplified production, the microballoon of namely first modifying with streptavidin prepares single stranded DNA 11 and 12, add again sequencing primer 13 to anneal with it, primer 13 can be corresponding with the partial sequence in sequence B, namely 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 successively respectively different dNTP to carry out burnt sequencing reaction during order-checking, obtain in 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 respectively paternal and maternal) usually, every type corresponding to a kind of template, namely two kinds of templates (target to be measured) only have a base sequence difference, these two kinds of templates can be corresponded respectively to TargetA and TargetB in 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 homozygote, only have 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 sequence B, to realize the same tense marker in a plurality of SNP site.
CNV (copy number variation) measures: when measuring copy number changes, must (namely there is no the variation of copy number) take one section sequence as basis of reference and measure the actual copy number of unknown template.Can be with the Target A in Fig. 1 as reference, Target B is target to be measured.If the sequence B 1 on Fig. 1 middle probe and B2 are used for different target to be measured on the marker gene group, in measurement result, the relative height at two peaks is the changing conditions of two testing gene fragment copy numbers.
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 substantially constant, can regard constant basis as.During mensuration, can be with the Target A in Fig. 1 as reference (house-keeping gene), Target B is target (testing gene).If the sequence B 1 on Fig. 1 middle probe and B2 are used for the marker gene kind, in measurement result, the relative height at two peaks is the relative expression quantity (being the template amount) of marker gene.
Embodiment 2: the method that the sequence label of a plurality of targets to be measured is decoded simultaneously
Method And Principle: when the dyestuff of employing different colours carries out mark to different targets to be measured, be subjected to the restriction of kind of dyes 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 measuring sequence, must use the isolation 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 isolation, measure when having realized a plurality of target like this.
In Fig. 2, use CAT, GAT, TCT, TGT, TAC, TAG is six kinds of targets of mark respectively, when adding dCTP, only have the sequence label corresponding with target 1 to go out peak (left side pentagram in Fig. 2); When adding dGTP equally, only have the sequence label corresponding with target 2 to go out peak (left side cross star in Fig. 2); Then add dTTP (isolation is used), except target 1 and 2, extension has all occured in all the other four kinds of targets, can obviously find out the peak of T in Fig. 2; When adding dCTP again, only have the sequence label corresponding with target 3 to go out peak (pentagram in the middle of in Fig. 2); When adding dGTP equally, only have the sequence label corresponding with target 4 to go out peak (cross star in the middle of in Fig. 2); Then add dATP (isolation is used), except target 3 and 4, extension has all occured in all the other four kinds of targets, can obviously find out the peak of A in Fig. 2; When adding dCTP again, only have the sequence label corresponding with target 5 to go out peak (the right pentagram in Fig. 2); When adding dGTP equally, only have the sequence label corresponding with target 6 to go out peak (the right cross star in Fig. 2).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 in theory infinitely a plurality of.
Embodiment 3: the mensuration of the multiple single base polymorphisms relevant to diabetes
1. experiment material
Reagent: genomic dna, by extracting in patient's blood sample; Ampligase (EPICENTRE); 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 sample adding liquid and ethidium bromide (EB) are Sangong biotech firm product.Dynabeads M-280-streptavidin (Dynal AS, Oslo, Norway), 1 * B﹠amp; 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, Suzhou safe and sound technology in the sky 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 to diabetes in 10 μ l reaction systems, it is respectively the C1431T site of PPAR gene, the C2821T site of PPAR gene, the Pro12Ala 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 for probe mixture, 4*3=12 pair altogether, every kind of 50fmol, the preparation method is referring to embodiment 1), Ampligase 1U, 20mM Tris-HCl (pH8.3), 25mM KCl, 10mM MgC12, 0.5mM NAD, 0.01%Triton X-100.Reaction conditions is: 98 ℃ of denaturation 5min; Then 95 ℃ of sex change 1min, connect 4min at 60 ℃ of annealing, carry out 10 circulations.
Polymerase chain reaction: contain primer (200nM) in 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 denaturation 5min; Then with 95 ℃ of 30s, 60 ℃ of 1min, carry out 35 circulations.
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 appropriate 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, more once uses afterwards B﹠amp with the 0.1mol/LNaOH washing; 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, then 55 ℃ of annealing 3min.Add sequencing reaction liquid in sequencing template, add successively dGTP, dCTP and dTTP to carry out sequencing reaction.
3. experimental result
Measured simultaneously four kinds of SNP sites relevant to diabetes in a pipe, Fig. 3 is the partial results of measuring three typical genomic dna samples, i.e. the measurement result in the C1431T site of PPAR gene.When the peak that only has the wild-type of representing in the mensuration collection of illustrative plates occurred, the C1431T that shows sample to be tested PPAR gene was wild-type; Except the peak that the wild-type of representing is arranged, represent that in addition the peak of mutant occurs in measuring collection of illustrative plates, show that the C1431T site of sample to be tested PPAR gene is heterozygote; When only having the peak of the mutant of representing in measuring collection of illustrative plates, the C1431T that shows sample to be tested PPAR gene is mutant.
Embodiment 4: the sequence label method is used for the mensuration of gene expression amount difference
The present embodiment adopts sequence label method of the present invention to investigate the expression of 5 genes (C-myc, CD44v6, H-ras, COX-2, RAB5A) in Colorectal Carcinoma, take house-keeping gene beta-actin as reference.Sequence label in 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, then 10min deactivation in the water-baths of 70 degree will be measured.Add mixed probe (6 pairs) (every kind of 5fmol) to 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, 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 the practical measurement result must be deducted this background.Result shows that six kinds of tumor-related genes all have expression in large bowel cancer, and the expression amount of COX-2 gene is the highest, but the expression amount of all genes is all low than house-keeping gene.
Sequence table
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＜120〉a kind of method of utilizing the sequence label quantitatively measuring nucleic acid
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ccccacttct tgttctctca t 21
Claims (4)
1. method of utilizing sequence label technology quantitatively measuring nucleic acid, comprising the following steps: that ligation mark determined nucleic acid, amplified reaction increase ligation product amount, sequencing reaction identification and be connected connects sequence label on reaction product, it is characterized in that containing in 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 linking probe;
Wherein:
Sequence label refers to have the DNA fragmentation of Different Alkali basic sequence;
Ligation refers to occur with the sequence of same determined nucleic acid the reaction of complementary a pair of adjacent linking probe generation under the effect of ligase enzyme:
Linking probe one end contains one section sequence with the complementation of determined nucleic acid template, and the other end contains one section sequence not complementary with the determined nucleic acid template;
The sequence not complementary with the determined nucleic acid template comprises one section PCR primer specificity sequence at the probe that does not contain sequence label, can also comprise one section restriction endonuclease specificity site sequence; The sequence not complementary with the determined nucleic acid template comprises one section PCR primer specificity sequence and sequence label at the probe that contains sequence label, can also comprise one section restriction endonuclease specificity site sequence;
After a pair of adjacent linking probe referred to two probes and same determined nucleic acid molecular hybridization, wherein 5 ' end of 3 ' of linking probe end and another linking probe was closely adjacent;
Sequencing reaction refers to measure based on bioluminescence reaction the sequencing technologies of the pyrophosphate salt of primer extension generation.
2. method according to claim 1, is characterized in that ligation refers in single tube many to the simultaneous ligation of adjacent linking probe.
3. method according to claim 1, is characterized in that restriction endonuclease refers to IIS type restriction endonuclease; The PCR primer refers to the general primer for the amplification connection product corresponding with multiple nucleic acids.
4. method according to claim 1, is characterized in that sequencing reaction refers to the sequencing reaction that adopts dNTP to carry out, or the sequencing reaction that adopts ddNTP to carry out.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1250483A (en) * | 1997-01-15 | 2000-04-12 | 布拉克斯集团有限公司 | Mass label linked hybridisation probes |
| 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 |
| CN1553953A (en) * | 2000-07-07 | 2004-12-08 | ά�������＼����˾ | Real-time sequence determination |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1250483A (en) * | 1997-01-15 | 2000-04-12 | 布拉克斯集团有限公司 | Mass label linked hybridisation probes |
| CN1553953A (en) * | 2000-07-07 | 2004-12-08 | ά�������＼����˾ | 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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