WO1999047700A1 - Procede et dispositif de detection d'une sequence nucleotidique - Google Patents
Procede et dispositif de detection d'une sequence nucleotidique Download PDFInfo
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- WO1999047700A1 WO1999047700A1 PCT/DE1999/000725 DE9900725W WO9947700A1 WO 1999047700 A1 WO1999047700 A1 WO 1999047700A1 DE 9900725 W DE9900725 W DE 9900725W WO 9947700 A1 WO9947700 A1 WO 9947700A1
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- WIPO (PCT)
- Prior art keywords
- molecule
- primer
- microtiter plate
- fluorophoric
- bound
- Prior art date
Links
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- 238000012546 transfer Methods 0.000 claims abstract description 8
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- 238000003786 synthesis reaction Methods 0.000 claims description 18
- WCKQPPQRFNHPRJ-UHFFFAOYSA-N 4-[[4-(dimethylamino)phenyl]diazenyl]benzoic acid Chemical compound C1=CC(N(C)C)=CC=C1N=NC1=CC=C(C(O)=O)C=C1 WCKQPPQRFNHPRJ-UHFFFAOYSA-N 0.000 claims description 15
- -1 polycarbene Polymers 0.000 claims description 11
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 claims description 10
- BZTDTCNHAFUJOG-UHFFFAOYSA-N 6-carboxyfluorescein Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C11OC(=O)C2=CC=C(C(=O)O)C=C21 BZTDTCNHAFUJOG-UHFFFAOYSA-N 0.000 claims description 7
- 230000000295 complement effect Effects 0.000 claims description 7
- SJQRQOKXQKVJGJ-UHFFFAOYSA-N 5-(2-aminoethylamino)naphthalene-1-sulfonic acid Chemical compound C1=CC=C2C(NCCN)=CC=CC2=C1S(O)(=O)=O SJQRQOKXQKVJGJ-UHFFFAOYSA-N 0.000 claims description 5
- 230000003321 amplification Effects 0.000 claims description 5
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- 238000003199 nucleic acid amplification method Methods 0.000 claims description 5
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- 239000004417 polycarbonate Substances 0.000 claims description 5
- WGTODYJZXSJIAG-UHFFFAOYSA-N tetramethylrhodamine chloride Chemical compound [Cl-].C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C(O)=O WGTODYJZXSJIAG-UHFFFAOYSA-N 0.000 claims description 5
- COCMHKNAGZHBDZ-UHFFFAOYSA-N 4-carboxy-3-[3-(dimethylamino)-6-dimethylazaniumylidenexanthen-9-yl]benzoate Chemical compound C=12C=CC(=[N+](C)C)C=C2OC2=CC(N(C)C)=CC=C2C=1C1=CC(C([O-])=O)=CC=C1C(O)=O COCMHKNAGZHBDZ-UHFFFAOYSA-N 0.000 claims description 4
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 4
- 150000007523 nucleic acids Chemical group 0.000 claims description 4
- 229920000049 Carbon (fiber) Polymers 0.000 claims description 3
- RUOKPLVTMFHRJE-UHFFFAOYSA-N benzene-1,2,3-triamine Chemical compound NC1=CC=CC(N)=C1N RUOKPLVTMFHRJE-UHFFFAOYSA-N 0.000 claims description 3
- 239000004917 carbon fiber Substances 0.000 claims description 3
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- 238000012545 processing Methods 0.000 claims description 3
- 108090000790 Enzymes Proteins 0.000 claims description 2
- 102000004190 Enzymes Human genes 0.000 claims description 2
- GMRJUBWVJSMUQV-UHFFFAOYSA-N 2,3,4-triethylthiophene Chemical compound CCC1=CSC(CC)=C1CC GMRJUBWVJSMUQV-UHFFFAOYSA-N 0.000 claims 1
- MAVVDCDMBKFUES-UHFFFAOYSA-N 2,3,4-trimethylthiophene Chemical compound CC1=CSC(C)=C1C MAVVDCDMBKFUES-UHFFFAOYSA-N 0.000 claims 1
- 230000002123 temporal effect Effects 0.000 claims 1
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- 238000003752 polymerase chain reaction Methods 0.000 description 32
- 108020004414 DNA Proteins 0.000 description 18
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- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical group O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 4
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- 108090000623 proteins and genes Proteins 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical group N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 108010006785 Taq Polymerase Proteins 0.000 description 2
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- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 2
- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N coumarin Chemical compound C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 2
- 238000002073 fluorescence micrograph Methods 0.000 description 2
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- 238000002515 oligonucleotide synthesis Methods 0.000 description 2
- 239000002953 phosphate buffered saline Substances 0.000 description 2
- 229940104230 thymidine Drugs 0.000 description 2
- JCYPECIVGRXBMO-UHFFFAOYSA-N 4-(dimethylamino)azobenzene Chemical compound C1=CC(N(C)C)=CC=C1N=NC1=CC=CC=C1 JCYPECIVGRXBMO-UHFFFAOYSA-N 0.000 description 1
- NJYVEMPWNAYQQN-UHFFFAOYSA-N 5-carboxyfluorescein Chemical group C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C21OC(=O)C1=CC(C(=O)O)=CC=C21 NJYVEMPWNAYQQN-UHFFFAOYSA-N 0.000 description 1
- ZMERMCRYYFRELX-UHFFFAOYSA-N 5-{[2-(iodoacetamido)ethyl]amino}naphthalene-1-sulfonic acid Chemical compound C1=CC=C2C(S(=O)(=O)O)=CC=CC2=C1NCCNC(=O)CI ZMERMCRYYFRELX-UHFFFAOYSA-N 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 108010000521 Human Growth Hormone Proteins 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
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- 229940079593 drug Drugs 0.000 description 1
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- 230000000694 effects Effects 0.000 description 1
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- IINNWAYUJNWZRM-UHFFFAOYSA-L erythrosin B Chemical compound [Na+].[Na+].[O-]C(=O)C1=CC=CC=C1C1=C2C=C(I)C(=O)C(I)=C2OC2=C(I)C([O-])=C(I)C=C21 IINNWAYUJNWZRM-UHFFFAOYSA-L 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- DLBFLQKQABVKGT-UHFFFAOYSA-L lucifer yellow dye Chemical compound [Li+].[Li+].[O-]S(=O)(=O)C1=CC(C(N(C(=O)NN)C2=O)=O)=C3C2=CC(S([O-])(=O)=O)=CC3=C1N DLBFLQKQABVKGT-UHFFFAOYSA-L 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
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- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- MPLHNVLQVRSVEE-UHFFFAOYSA-N texas red Chemical compound [O-]S(=O)(=O)C1=CC(S(Cl)(=O)=O)=CC=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 MPLHNVLQVRSVEE-UHFFFAOYSA-N 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
Definitions
- the invention relates to a method according to the preamble of claim 1. It also relates to a microtiter plate and a kit for performing the method.
- An amplification method is known from WO93 / 09250, in which a first primer is bound to a first phase.
- a second primer is labeled with a fluorophore dye. If a nucleotide sequence to be detected is present, the labeled second primer accumulates on the solid phase. - In order to be able to recognize a sufficiently discriminating signal on the solid phase, it is necessary to carry out a washing step after the PCR. This step requires additional work. Contamination can also occur.
- the object of the present invention is to eliminate the disadvantages of the prior art.
- a simple and inexpensive method with improved sensitivity and less contamination probability is to be specified.
- the concentration of the nucleotide sequence to be detected should be determinable as efficiently as possible.
- At least one of the fluorophoric molecules is bound to the surface of a solid phase.
- the method allows a qualitative and quantitative determination of the nucleotide sequence to be detected.
- simple fluorescence measurement in particular online detection, is possible.
- the process can be carried out simply and inexpensively because washing steps which increase the risk of contamination can be dispensed with.
- a first primer is bound to the solid phase.
- the first fluorophoric molecule may be bound to the solid phase via the first primer.
- the first primer advantageously has a hairpin loop, and the first fluorophoric molecule is bound to one loop section and the second fluorophoric molecule opposite to the other loop section at a distance that enables the interaction.
- the interaction is expediently eliminated by hybridization with a complementary strand complementary to the first primer or by a synthesis taking place on the first primer. The probability of contamination is further reduced by the aforementioned procedure.
- the second fluorophoric molecule can also be bound to a second primer.
- a second primer is in solution.
- the first and second primers are advantageously hybridized in such a way that the interaction is generated.
- the distance between the first and second fluorophoric molecules is preferably 2 to 12 nucleotides.
- the solid phase can contain a, preferably electrically conductive, plastic, for example a polycarbonate, polycarbene, trimethylthiopene and / or triaminobenzene and / or carbon fibers. It has proven to be particularly advantageous that the solid phase is a microtiter plate.
- the first molecule is an acceptor group and the second fluorophoric molecule is a donor group.
- the acceptor group can be a 6-carboxy-tetramethyl-rhodamine and the donor group can be a 6-carboxy-fluorescein.
- Other suitable donor / acceptor pairs are shown in the table below:
- IAEDANS 5 - ((((2-fluorescein iodoacyl) amino) ethyl) amino) nap hathalene-isulonic acid)
- first and second fluorophoric molecules can be interchanged.
- the first or second fluorophoric molecule can be replaced by a quencher, preferably formed from 4- [4 '-dimethylaminophenylazo] benzene acid.
- the fluorescence can be detected by means of a fluorometer connected to a data processing device, the concentration of the nucleotide sequence to be detected being determined from the change in the fluorescence intensity over time.
- the second derivative of the fluorescence intensity over the number of amplification cycles carried out is preferably used as the reference point.
- a microtiter plate with a top side having several trough-shaped depressions is provided for carrying out the method according to the invention, to which the first molecule is bound.
- a first primer can be bound to the top side, the first molecule advantageously being bound to the surface via the first primer.
- the first primer has a hairpin loop, and the first molecule is bound to a loop section and the second molecule opposite to a second loop section at a distance that enables the interaction.
- a kit with a microtiter plate according to the invention and a primer provided with a second molecule is provided.
- 7b the particle according to FIG. 7a in a fluorescence microscope image
- 7c shows a particle bound to a second primer after a PCR without template DNA in a dark field image
- a first primer P1 is bound to the top inside a cavity of a microtiter plate M made of polycarbonate or polypropylene.
- the microtiter plate M can contain a controlled resistance heater. It can also be a resistance heating element itself.
- a first fluorophore molecule F1 is bound to the first primer P1.
- the nucleic acid sequence N to be detected contained in a target DNA and the further components necessary for carrying out a polymerase chain reaction (PCR) or ligase chain reaction (LCR) are pipetted into the cavities. These contain in particular a second primer P2 with a second fluorophore molecule F2 bound to it.
- PCR polymerase chain reaction
- LCR ligase chain reaction
- the target DNA is denatured by increasing the temperature, i.e. separated into a strand S and a counter strand G.
- the temperature is then reduced to 50 to 60 °.
- the strand S binds to the first primer P1 with a complementary sequence section.
- the counter strand G binds to the second primer P2 in the liquid.
- the missing sequence section is then synthesized using a Taq DNA polymerase. Then the temperature is raised to 94 ° C., so that the synthesis strands containing the fluorophoric molecules F1, F2 as single strands, namely as synthesis strand SSI and as synthesis counter strand SGI, in the 8th
- the second fluorophoric molecule F2 can also be incorporated into the synthesis strand SSI bound to nucleotides or a further nucleic acid sequence.
- the temperature is reduced to 50 to 60 °.
- the synthesis strand SSI and the synthesis counter strand SGI hybridize so that the first F1 and the second fluorophore molecule F2 are at a distance of 6 to 12 nucleotides.
- Fig. 2 shows this schematically.
- the first fluorophoric molecule F1 which is designed as a donor
- the second fluorophoric molecule F2 which acts as an acceptor.
- an increased fluorescence is observed on the second fluorophoric molecule F2.
- the fluorescence is detected using a fluorometer. The detected values are forwarded to a data processing system.
- the first primer P1 can also have a hairpin loop, the first fluorophore molecule F1 being bound to a first loop section and a quencher opposite to a loop section at a distance which enables the interaction.
- the hairpin loop is closed, the interaction causes the fluorescence to be quenched.
- the hairpin loop is opened by hybridization with a counter strand G complementary to the first primer P1 or by a synthesis taking place on the first primer P1.
- the interaction between the fluorophore molecule and the quencher is broken. When the fluorophoric molecules are excited, fluorescence occurs.
- the next PCR cycle is then initiated by increasing the temperature. This leads to a further increase in the Synthesis strand SSI and the synthesis counter strand SGI and consequently to an increase in the fluorescence intensity.
- the change in the fluorescence intensity over the number of PCR or LCR cycles is a measure of the initial concentration of the target DNA: the more target DNA is contained in a sample, the faster the fluorescence intensity increases.
- a microtiter plate M made of polycarbonate or polypropylene is used to carry out the aforementioned method.
- the first primer P1 is bound to a polypropylene surface with its 5 'end via a linker, which preferably consists of 6 CH 2 groups.
- the first primer P1 is bound to the polypropylene surface by the method of Weiler-J. and Hoheisel-JD. (Anal. Biochem., 1996; 243 (2): 218-27).
- the first fluorophore molecule F1 is directly attached to the solid phase, i.e. the top of the microtiter plate M, bound.
- the first primer P1 is bound to the solid phase in the vicinity of the first fluorophoric molecule F1.
- After a hybridization of the synthesis strands SSI or the synthesis counterstrands SGI when there is an excitation, there is a radiation-free energy transition from the first fluorophoric molecule F1 (donor) to the second fluorophore molecule F2 (acceptor) and fluorescence there (FIG. 5).
- FIG. 6 shows the fluorescence of PCR products of PCR with 3'-fluorophore-bearing primers.
- the fluorescence of the PCR product is at an excitation wavelength of 496 im 10
- the sample "PCR without template” is a PCR approach without HGH template DNA after 25 cycles.
- the sample “PCR with template” is a PCR approach with HGH template DNA after 25 cycles.
- the right column shows the PCR approach with template DNA, but without performing temperature cycles.
- Example 1 Fluorescence energy transfer in PCR products from 3'-fluorophore-bearing primers
- Two primers are synthesized which were labeled with fluorophoric groups in the region of the 3 'end.
- a first primer with a length of 23 bases has the following sequence:
- the thymidine at position 4 with respect to the 3 'end (printed in bold in the sequence) is labeled with 6-carboxyfluorescein (6-FAM).
- 6-carboxyfluorescein (6-FAM).
- the FAM group is linked via the amino group of the dT-C2-NH2 incorporated during the oligonucleotide synthesis.
- the thymidine at position 3 with respect to the 3 'end (printed in bold in the sequence) is marked with carboxymethylrhodamine (TAMRA).
- TAMRA carboxymethylrhodamine
- the TAMRA group is linked via the a ino group of the dT-C2-NH2 incorporated during oligonucleotide synthesis.
- the second primer is labeled with a biotin group at the 5 'end.
- the synthesis scale is 0.2 ⁇ mol.
- the primers are cleaned by HPLC.
- the sequences of the primers are located immediately adjacent to a sequence section of the human growth hormone gene (HGH gene).
- HGC 5'-ACCAGGAGTTTGTAAGCTCTTGG-GGAATGGGTGCGCATCAGG-3 '3' -TGGTCCTCAAACATTCGAGAACC-CCTTACCCACGCGTAGTCC-5 '
- Second primer 3 * -CCTTACCCACGCGTAGTCC-Biotin-5 '
- the first and second primers are reacted in a PCR using a template DNA covering the sequence portion of the HGH gene.
- the PCR is carried out in a total volume of 50 ⁇ l, each with 0.5 ⁇ M primer, 2 units of Taq DNA polymerase and 1 ⁇ l HGH gene (10ng) in the corresponding PCR buffers (all solutions and enzymes from Boehringer, Mannheim). 25 cycles with an annealing temperature of 66 ° C (45 seconds), elongation temperature of 72 ° C (45 seconds) and a denaturation temperature of 94 ° C (30 seconds) are carried out. 12
- the same PCR is carried out, leaving out the template DNA.
- the PCR mixture is left at 4 ° C.
- the PCR forms a PCR product in which the fluorophores of the first and second primers are arranged at a distance of a few bases on the strands of opposite polarity:
- the fluorescence is determined in a fluorescence spectrometer with an excitation of 496nm (+/- 10nm) and an emission of 576n (+/- 10nm).
- the fluorescence of the TAMRA group is increased by the PCR (FIG. 6). This increase in fluorescence indicates the formation of the expected PCR product. 13
- Example 2 The same primers described in Example 1 are used for the PCR with 3 '-labelled and immobilized primers.
- the 5 '-biotinylated second primer according to Example 1 is bound by the PCR to streptavidin-coated, super-paramagnetic particles with a size of approximately 2.8 ⁇ m in diameter (M-280 Dynabeads, Dynal, Hamburg).
- the particles (10 ⁇ g / ⁇ l; 6.7 x 10 8 particles / ml suspended in phosphate buffered saline (PBS) pH 7.4 with B / W buffer (10 mm Tris-Cl, ImM EDTA, 2M NaCl) pH 7 , 5 and brought to a concentration of 5 ⁇ g / ⁇ l in B / W buffer
- B / W buffer 10 mm Tris-Cl, ImM EDTA, 2M NaCl
- TE lOmM TrisCl, 0.2mM EDTA pH8
- the PCR according to Example 1 is carried out with the second primer bound to the supermagnetic particle.
- 1 ⁇ l of the suspension of particle-bound primer-2 is used instead of the free second primer.
- the particles are washed several times in TE and analyzed in a fluorescence microscope. The attachment of the 6-FAM-labeled first primer to the particles is examined. 7A shows the fluorescence of the particles after completion. A fluorescence of the particles can be observed in the PCR approach shown in FIG. 7B. 14
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- General Health & Medical Sciences (AREA)
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Abstract
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2000536883A JP2002506654A (ja) | 1998-03-18 | 1999-03-16 | ヌクレオチド配列を検出するための方法と装置 |
EP99919084A EP1064407A1 (fr) | 1998-03-18 | 1999-03-16 | Procede et dispositif de detection d'une sequence nucleotidique |
CA002323075A CA2323075A1 (fr) | 1998-03-18 | 1999-03-16 | Procede et dispositif de detection d'une sequence nucleotidique |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE19811729A DE19811729C2 (de) | 1998-03-18 | 1998-03-18 | Verfahren und Vorrichtung zum Nachweis einer Nukleotidsequenz |
DE19811729.9 | 1998-03-18 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1999047700A1 true WO1999047700A1 (fr) | 1999-09-23 |
Family
ID=7861305
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/DE1999/000725 WO1999047700A1 (fr) | 1998-03-18 | 1999-03-16 | Procede et dispositif de detection d'une sequence nucleotidique |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP1064407A1 (fr) |
JP (1) | JP2002506654A (fr) |
CA (1) | CA2323075A1 (fr) |
DE (1) | DE19811729C2 (fr) |
WO (1) | WO1999047700A1 (fr) |
Cited By (5)
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DE19960076C2 (de) * | 1999-12-13 | 2002-12-05 | November Ag Molekulare Medizin | Verfahren und Vorrichtung zum Nachweis und zur Quantifizierung von Biomolekülen |
WO2013055647A1 (fr) | 2011-10-11 | 2013-04-18 | Enzo Life Sciences, Inc. | Colorants fluorescents |
US9068948B2 (en) | 2002-03-12 | 2015-06-30 | Enzo Life Sciences, Inc. | Processes for detection of nucleic acids |
US9353405B2 (en) | 2002-03-12 | 2016-05-31 | Enzo Life Sciences, Inc. | Optimized real time nucleic acid detection processes |
CN112996899A (zh) * | 2018-11-09 | 2021-06-18 | 横河电机株式会社 | 核酸序列检测用装置 |
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DE10111420A1 (de) * | 2001-03-09 | 2002-09-12 | Gnothis Holding Sa Ecublens | Bestimmung von Analyten durch Fluoreszenz-Korrelationsspektroskopie |
JP4710283B2 (ja) * | 2004-09-08 | 2011-06-29 | 富士レビオ株式会社 | 核酸配列の増幅法および検出法 |
CN102851369B (zh) | 2006-12-13 | 2015-01-21 | 卢米耐克斯公司 | 用于实时pcr多重分析的系统和方法 |
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US9068948B2 (en) | 2002-03-12 | 2015-06-30 | Enzo Life Sciences, Inc. | Processes for detection of nucleic acids |
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US9316587B2 (en) | 2002-03-12 | 2016-04-19 | Enzo Life Sciences, Inc. | Processes for quantitative or qualitative detection of single-stranded or double-stranded nucleic acids |
US9353405B2 (en) | 2002-03-12 | 2016-05-31 | Enzo Life Sciences, Inc. | Optimized real time nucleic acid detection processes |
US9416153B2 (en) | 2011-10-11 | 2016-08-16 | Enzo Life Sciences, Inc. | Fluorescent dyes |
US10106573B2 (en) | 2011-10-11 | 2018-10-23 | Enzo Life Sciences, Inc. | Fluorescent dyes and methods of use thereof |
WO2013055647A1 (fr) | 2011-10-11 | 2013-04-18 | Enzo Life Sciences, Inc. | Colorants fluorescents |
EP3747877A1 (fr) | 2011-10-11 | 2020-12-09 | Enzo Life Sciences, Inc., c/o Enzo Biochem, Inc. | Colorants fluorescents |
US10875886B2 (en) | 2011-10-11 | 2020-12-29 | Enzo Life Sciences, Inc. | Fluorescent dyes and methods of use thereof |
US11939350B2 (en) | 2011-10-11 | 2024-03-26 | Enzo Life Sciences, Inc. | Fluorescent dyes and methods of use thereof |
CN112996899A (zh) * | 2018-11-09 | 2021-06-18 | 横河电机株式会社 | 核酸序列检测用装置 |
CN112996899B (zh) * | 2018-11-09 | 2024-07-05 | 横河电机株式会社 | 核酸序列检测用装置 |
Also Published As
Publication number | Publication date |
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JP2002506654A (ja) | 2002-03-05 |
EP1064407A1 (fr) | 2001-01-03 |
CA2323075A1 (fr) | 1999-09-23 |
DE19811729C2 (de) | 2000-05-18 |
DE19811729A1 (de) | 1999-09-23 |
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