CA2323075A1 - Method and device for detecting a nucleotide sequence - Google Patents
Method and device for detecting a nucleotide sequence Download PDFInfo
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- CA2323075A1 CA2323075A1 CA002323075A CA2323075A CA2323075A1 CA 2323075 A1 CA2323075 A1 CA 2323075A1 CA 002323075 A CA002323075 A CA 002323075A CA 2323075 A CA2323075 A CA 2323075A CA 2323075 A1 CA2323075 A1 CA 2323075A1
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- 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
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Abstract
The invention relates to a method for detecting a nucleotide sequence using fluorescence. According to the inventive method, an interaction made possible by direct energy transfer or without radiation occurs between a first (F1) and a second molecule (F2) in the presence of a nucleotide sequence (N) that is to be detected. At least one of the molecules (F1, F2) is bonded to the surface of a solid phase (M) in order to avoid contamination and to improve the sensitivity of the method.
Description
wo 99/47700 PCT/DE99/00725 Method and device for detecting a nucleotide sequence The invention relates to a method in accordance with the preamble of Claim 1. It also relates to a microtiter plate and a kit for carrying out the method.
US 4,996,143 and DE 195 81 489 Tl disclose methods in which a first and a second primer are bound to the nucleotide sequence to be detected at a distance of 2 to 7 nucleotides. The first and the second primer are each provided with a fluorophoric .molecule. In the bound state, a radiation-free energy transfer from one fluorophoric molecule to the other is observed owing to the Forster effect. This causes a specific fluorescence. - The known method is not particularly sensitive.
US 5,607,834 discloses the use of a primer with a hair-pin loop for detecting a nucleotide sequence. In this case, fluorophoric molecule and a quencher are provided opposite each other on the loop sections of the hair-pin loop. The distance between the fluorophoric molecule and the quencher allow [sic] a radiation-free energy transfer which quenches the fluorescence.
However, when the primer hybridizes with a complementary strand, the hairpin is opened. The spatial relationship between the fluorophoric molecule and the quencher, which quenches a fluorescence, is altered. Thus, a fluorescence is observable.
W093/09250 discloses an amplification method in which a first primer is bound to a first phase. A second primer is labeled with a fluorophoric ,dye. When a nucleotide sequence to be detected is present, the labeled second primer accumulates on the solid phase. - In order to recognize a sufficiently discriminating signal on the solid phase, it is necessary to carry out a washing REPLACEMENT SHEET (RULE 26) step after the PCR. This step requires an additional effort. Moreover, contaminations may be introduced while carrying out this step.
The object of the present invention is to eliminate the disadvantages of the prior art; it is intended in particular to provide a method with improved sensitivity, where the possibility of contamination is reduced and which is simple and inexpensive to carry out. Moreover, the concentration of the nucleotide sequence to be detected is to be determined in as efficient a manner as possible.
This object is achieved by the features of claims 1 and 19. Expedient embodiments result from the features of claims 2 to 18 and 20 to 34.
In accordance with the invention, at least one of the fluorophoric molecules is bound to the surface of a solid phase. The method allows the nucleotide sequence to be detected to be determined qualitatively and quantitatively. A simple fluorescence measurement, in particular an online detection, is possible owing to the fact that the at least one fluorophoric molecule is bound to a solid phase. The method can be carried out in a simple and inexpensive manner since washing steps, which increase the risk of contamination, can be dispensed with.
In a particular embodiment, a first primer is bound to the solid phase. It is possible that the first fluorophoric molecule is bound to the solid phase via the first primer. In this case, the first primer advantageously has a hairpin loop, and the first fluorophoric molecule is bound to the one loop section and the second fluorophoric molecule opposite to the other loop section at a distance which allows the REPLACEMENT SHEET (RULE 26) interaction to take place. The interaction is eliminated expediently by hybridization with a complementary strand which is complementary to the first primer or by a synthesis which takes place on the first primer. The above-described procedure further reduces the possibility of contamination.
In a further embodiment of the method, the second fluorophoric molecule can also be bound to a second primer. However, it is also possible to incorporate nucleotides provided with the second fluorophoric molecule, or a further nucleic acid sequence, into a synthesis strand. The second primer is in solution.
After amplification and denaturation, the first and the second primer are advantageously hybridized in such a manner that the interaction is generated. The distance between the first and the second fluorophoric molecule in the hybridized state is preferably 2 to 12 nucleotides. The above variant of the method is particularly sensitive.
The solid phase can comprise a polymer which is preferably electroconductive, for example a polycarbonate, polycarbene, trimethylthiopene and/or triaminobenzene and/or carbon fibers. It has proved to be especially advantageous for the solid phase to be a microtiter plate.
In a further feature of an embodiment, the first molecule is an acceptor group and the second fluorophoric molecule a donor group. The acceptor group can be a 6-carboxytetramethylrhodamine and the donor group a 6-carboxyfluorescein. Other suitable donor/acceptor pairs can be seen from the table which follows:
REPLACEMENT SHEET (RULE 26) Donor Acceptor Fluorescein Fluorescein Fluorescein Tetramethylrhodamine IAEDANS (= 5-((((2- Fluorescein iodacyl) amino) ethyl) amino) -naphathalene-lsulon [sic]
acid) EDANS (=5-((2-aminomethyl)- DABCYL [sic] (4-dimethyl-amino)naphthalene-1- aminoazo-benzene-4'-sulfonic acid) sulfoylo chloride) [sic]
BODOPY [sic] FL BODIPY FL
Naturally, it is possible to swap the first and the second fluorophoric molecule. In a further feature of an embodiment, the first or second fluorophoric molecule can be replaced by a quencher, preferably a quencher formed by 4-[4'-dimethylaminophenylazo]benzoic acid.
Suitable quencher/fluorophore pairs can be seen from the table which follows:
Quencher Fluorophore DABCYL [sic] Coumarin DABCYL [sic] EDANS
DABCYL [sic] Fluorescein DABCYL [sic] Lucifer Yellow DABCYL [sic] Bodipy DABCYL [sic] Eosin DABCYL [sic] Tetramethylrhodamine DABCYL [sic] Texas Red DABCYL [sic] Erythrosin To determine the concentration of the nucleotide sequence to be detected, the fluorescence can be recorded by means of a fluorometer connected to a data REPLACEMENT SHEET (RULE 26) processing system, the concentration of the nucleotide sequence to be detected being determined from the change of the fluorescence intensity over time. The reference point used is preferably the second derivative of the fluorescence intensity over the number of the amplification cycles carried out.
In accordance with the solution with regard to the device, a microtiter plate is provided for carrying out the method according to the invention with an upper face which is provided with a plurality of well-shaped recesses and to which the first molecule is bound. A
first primer may be bound to the upper face, the first molecule advantageously being bound to the surface via the first primer. In a further embodiment, the first primer has a hairpin loop, and the first molecule is bound to one loop section and the second molecule opposite to a second loop section at a distance which allows the interaction to take place.
In a further embodiment with regard to the device, a kit is provided with a microtiter plate according to the invention and with a primer provided with a second molecule.
The method according to the invention is illustrated in greater detail with reference to the drawing. In this drawing, Fig. 1 shows the pairing of the strand and the complementary strand of a target DNA with a first and a second primer, Fig. 2 the hybridization of the primers synthesized, Fig. 3 the excitation of the fluorophoric molecules, REPLACEMENT SHEET (RULE 26) Fig. 4 the pairing of the strand and the complementary strand of a target DNA in a further variant of the method, Fig. 5 the excitation of the fluorophoric molecules in accordance with the variant of the method in Fig. 4, Fig. 6 the fluorescence of a detector nucleotide with and without nucleotide label, Fig. 7a a particle bound to a second primer following PCR with template DNA in a dark field image, Fig. 7b the particle of Fig. 7a in a fluorescence micrograph, Fig. 7c a particle bound to a second primer following PCR without template DNA in a dark field image, and Fig. 7d the particle of Fig. 7c in a fluorescence micrograph.
In Fig. l, a first primer Pl is bound to the upper face within a cavity of a microtiter plate M made of polycarbonate or polypropylene. The microtiter plate M
may contain a controlled resistance heating. It may also be an element of a resistance heating itself. A
first fluorophoric molecule Fl is bound to the first primer P1.
The nucleic acid sequence N to be detected, which is present in a target DNA, and the further components required for carrying out a polymerase chain reaction (PCR) or ligase chain reaction (LCR) are pipetted into the cavities. The latter comprise in particular a REPLACEMENT SHEET (RULE 26) _ 7 _ second primer P2 with a second fluorophoric molecule F2 bound thereto. The target DNA is denatured, i.e.
separated into a strand S and a complementary strand C.
The temperature is then reduced to 50 to 60° [sic]. The strand S binds with a complementary sequence segment to the first primer P1. The complementary strand G binds to the second primer P2 which is present in the fluid.
Then, the sequence segment which is missing in each case is synthesized by means of a Taq DNA polymerase.
The temperature is then raised to ~94°C, so that the synthesis strands comprising the fluorophoric molecules Fl, F2 are present in the fluid as single strands viz as synthesis strand SSl and as synthesis complementary strand SC1. The second fluorophoric molecule F2 may also be incorporated into the synthesis strand SSl in a form in which it is bound to nucleotides or a further nucleic acid sequence, instead of via the second primer P2. The temperature is reduced to 50 to 60° [sic] . The synthesis strand SS1 and the synthesis complementary strand SC1 hybridize, so that the first F1 and the second fluorophoric molecule F2 are present at a distance of 6 to 12 nucleotides. This is shown schematically in Fig. 2.
Upon excitation of the first fluorophoric molecule F1, which is designed as the donor, a radiation-free energy transfer to the second fluorophoric molecule F2, which acts as the acceptor, takes place. As a consequence, an increased fluorescence is observed on the second fluorophoric molecule F2. The fluorescence is detected by means of a fluorometer. The readings are transmitted to a data-processing system.
The first primer Pl may also exhibit a hairpin loop, the first fluorophoric molecule F1 being bound to a first loop section and a quencher being bound to a loop REPLACEMENT SHEET (RULE 26) _ g _ section opposite at a distance which allows the interaction to take place. When the hairpin loop is closed, the interaction causes the fluorescence to be quenched. As a result of hybridization with a complementary strand C which is complementary to the first primer P1 or by a synthesis which takes place on the first primer P1, the hairpin loop is opened up. The interaction between the fluorophoric molecule and the quencher is eliminated. Excitation of the fluorophoric molecules results in fluorescence.
Then, the next PCR cycle is started by raising the temperature. The synthesis strand SSl and the synthesis complementary strand SC1 are multiplied further, and, as a result, the fluorescence intensity is increased.
The change in fluorescence intensity over the number of PCR or LCR cycles is a measure for the initial concentration of the target DNA: the more target DNA a sample contains, the more rapidly the fluorescence intensity increases.
To carry out the abovementioned method, a microtiter plate M made of polycarbonate or polypropylene is used.
The first primer P1 is bound with its 5'-terminus in the well area to the upper face of the microtiter plate M to a polypropylene surface via a linker which is preferably composed of 6 CH2 groups. The first primer Pl is bound to the polypropylene surface by the method of Weiler-J. and Hoheisel-JD. (Anal.- [sic] Biochem., 1996; 243 (2) . 218-27).
Fig. 4 shows a further variant of the method. In this variant, the first fluorophoric molecule F1 is bound directly to the solid phase, i.e. the upper face of the microtiter plate M. The first primer P1 is bound to the solid phase in the vicinity of the first fluorophoric REPLACEMENT SHEET (RULE 26) molecule Fl. After hybridization of the synthesis strands SSl or the synthesis complementary strands SCl, excitation results in a radiation-free energy transfer from the first fluorophoric molecule F1 (donor) to the second fluorophoric molecule F2 (acceptor), where fluorescence results (Fig. 5).
Fig. 6 shows the fluorescence of PCR products of the PCR with primers which have 3'-fluorophores attached to them. The fluorescence of the PCR product has been measured in relative fluorescence units (RFU) at an excitation wavelength of 496 nm and an emission wavelength of 576 nm. The sample ~~PCR without template"
is a PCR preparation without HGH template DNA after 25 cycles . The sample '~PCR with template" is a PCR mix with HGH template DNA after 25 cycles. The column on the right shows the PCR mix with template DNA, but without temperature cycles having been carried out.
It can be seen clearly from Fig. 6 that the template can be detected readily with the aid of the method according to the invention, in particular without any need for washing steps.
Example l:
Fluorescence energy transfer in PCR products of primers which have 3'-fluorophores attached to them Two primers labeled with fluorophoric groups in the 3'-terminus zone are synthesized.
A first primer with a length of 23 bases has the following sequence:
5'-ACCAGGAGTTTGTAAGCTCTTGG-3'.
REPLACEMENT SHEET (RULE 26) wo 99/47700 PCT/DE99/00725 The thymidine in position 4 relative to the 3'-terminus (emboldened in the sequence) is labeled with 6-carboxyfluorescein (6-FAM). The FAM group is bound via the amino group of the dT-C2-NH2, which is incorporated during oligonucleotide synthesis.
A second primer with a length of 19 bases has the following sequence:
5'-biotin-CCTGATGCGCACCCATTCC-3' The thymidine in position 3 relative to the 3'-terminus (emboldened in the sequence) is labeled with carboxymehtylrhodamine [sic] (TAMRA). The TAMRA group is bound via the amino group of the dT-C2-NH2, which is incorporated during oligonucleotide synthesis. The second primer is labeled at the 5'-terminus with a biotin group.
The synthesis is carried out on a 0.2~mol scale. The primers are purified by HPLC. The sequences of the primers are in immediate vicinity of a sequence segment of the human growth hormone gene (HGH gene).
First primer: 5'-ACCAGGAGTTTGTAAGCTCTTGG-3' 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 which covers the sequence segment of the HGH gene. The PCR is carried out in a total volume of 501 with in each case 0.5~M primer, 2 units of Taq-DNA
polymerase and 1~1 of HGH gene (long) in the relevant PCR buffers (all solutions and enzymes from Boehringer, REPLACEMENT SHEET (RULE 26) 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.
As negative control, the same PCRs are carried out, but without the template DNA. As a further control, the PCR
mix is left at 4°C.
The PCR results in the formation of the PCR product in which the fluorophores of the first and second primers are arranged on the strands of opposite polarity at a distance of a few bases:
FAM
5'-ACCAGGAGTTTGTAAGCTCTTGG-GGAATGGGTGCGCATCAGG-3' 3'-TGGTCCTCAAACATTCGAGAACC-CCTTACCCACGCGTAGTCC-biotin-5' TAMRA
A suitable excitation of the 5-FAM group at 496nm results in a fluorescence energy transfer to the TAMRA
group which is located on the complementary strand and which has an emission maximum at 576nm.
To detect the formation of the PCR product and the fluorescence energy transfer, the fluorescence is determined in a fluorescence spectrometer at an excitation of 496nm(+/- lOnm) and an emission of 576 nm (+/- lOnm). Owing to the PCR, the fluorescence of the TAMRA group increases (Fig. 6). This increase in fluorescence shows the formation of the expected PCR
product.
REPLACEMENT SHEET (RULE 26) Example 2:
PCR with 3'-labeled and immobilized primers The primers which described in Example 1 are also used for the PCR with 3'-labeled and immobilized primers.
The 5'-biotinylated second primer in accordance with Example 1 is bound by the PCR to streptavidin-coated, superparamagnetic particles with a size of approx.
2.8 ~m in diameter (M-280 Dynabeads, Dynal, Hamburg). To this end, the particles (10~g/~1; 6.7 x 108 particles/ml suspended in phosphate-buffered saline (PBS) pH 7.4 are washed with B/W buffer (lOmM Tris-C1, 1mM EDTA, 2M
NaCl) ph [sic] 7.5 and brought to a concentration of 5 ~g/~1 in B/W buffer. 20~t1 of this suspension are treated with an equal volume of 50~M solution of primer-2 in distilled water. The suspension is incubated for 1 hour at room temperature with gentle shaking. Unbound primer is removed by washing the particles twice, first with 1001 of B/W buffer and then by washing with lOmM TrisCl, 0.2 mM EDTA pH8 (TE).
The particles are stored at 4°C in TE in a suspension of 10~g/~1.
The PCR with the second primer which is bound to the supermagnetic particle is carried out as in Example 1.
In contrast to Example 1, l~l of the suspension of the particle-bound primer-2 is employed instead of the free second primer. After the PCR, the particles are washed repeatedly with TE and analyzed on the fluorescence microscope. What is studied is the attachment of the 6-FAM-labeled first primer to the particles. Fig. 7A
demonstrates the fluorescence of the particles after conclusion. The PCR mix shown in Fig. 7B shows a fluorescence of the particles caused by the attachment of the FAM-labeled first primer to the particles. This fluorescence is not present in the control without template DNA (Fig. 7D).
REPLACEMENT SHEET (RULE 26) The symbols denote:
S Strand C Complementary strand P1 First primer P2 Second primer Fl First fluorophoric molecule F2 Second fluorophoric molecule SSl Synthesis strand SCl Synthesis complementary strand M Microtiter plate N Nucleotide sequence REPLACEMENT SHEET (RULE 26) SEQUENCE PROTOCOLS
<110> november AG Novus Medicatus Bertling Gesellschaft fur Molekolare [sic] Medizin <120> Method and device for detecting a nucleotide sequence <130> 390687ga5 <140>
<141>
<160> 4 <170> PatentIn Ver. 2.1 <210> 1 <211> 23 <212> DNA
<213> human <400> 1 accaggagtt tgtaagctct tgg 23 <210> 2 <211> 42 <212> DNA
<213> human <400> 2 accaggagtt tgtaagctct tggggaatgg gtgcgcatca gg 42 <210> 3 <211> 42 <212> DNA
<213> human REPLACEMENT SHEET (RULE 26) <400> 3 cctgatgcgc acccattccc caagagctta caaactcctg gt 42 <210> 4 <211> 19 <212> DNA
<213> human <400> 4 cctgatgcgc acccattcc 19 REPLACEMENT SHEET (RULE 26)
US 4,996,143 and DE 195 81 489 Tl disclose methods in which a first and a second primer are bound to the nucleotide sequence to be detected at a distance of 2 to 7 nucleotides. The first and the second primer are each provided with a fluorophoric .molecule. In the bound state, a radiation-free energy transfer from one fluorophoric molecule to the other is observed owing to the Forster effect. This causes a specific fluorescence. - The known method is not particularly sensitive.
US 5,607,834 discloses the use of a primer with a hair-pin loop for detecting a nucleotide sequence. In this case, fluorophoric molecule and a quencher are provided opposite each other on the loop sections of the hair-pin loop. The distance between the fluorophoric molecule and the quencher allow [sic] a radiation-free energy transfer which quenches the fluorescence.
However, when the primer hybridizes with a complementary strand, the hairpin is opened. The spatial relationship between the fluorophoric molecule and the quencher, which quenches a fluorescence, is altered. Thus, a fluorescence is observable.
W093/09250 discloses an amplification method in which a first primer is bound to a first phase. A second primer is labeled with a fluorophoric ,dye. When a nucleotide sequence to be detected is present, the labeled second primer accumulates on the solid phase. - In order to recognize a sufficiently discriminating signal on the solid phase, it is necessary to carry out a washing REPLACEMENT SHEET (RULE 26) step after the PCR. This step requires an additional effort. Moreover, contaminations may be introduced while carrying out this step.
The object of the present invention is to eliminate the disadvantages of the prior art; it is intended in particular to provide a method with improved sensitivity, where the possibility of contamination is reduced and which is simple and inexpensive to carry out. Moreover, the concentration of the nucleotide sequence to be detected is to be determined in as efficient a manner as possible.
This object is achieved by the features of claims 1 and 19. Expedient embodiments result from the features of claims 2 to 18 and 20 to 34.
In accordance with the invention, at least one of the fluorophoric molecules is bound to the surface of a solid phase. The method allows the nucleotide sequence to be detected to be determined qualitatively and quantitatively. A simple fluorescence measurement, in particular an online detection, is possible owing to the fact that the at least one fluorophoric molecule is bound to a solid phase. The method can be carried out in a simple and inexpensive manner since washing steps, which increase the risk of contamination, can be dispensed with.
In a particular embodiment, a first primer is bound to the solid phase. It is possible that the first fluorophoric molecule is bound to the solid phase via the first primer. In this case, the first primer advantageously has a hairpin loop, and the first fluorophoric molecule is bound to the one loop section and the second fluorophoric molecule opposite to the other loop section at a distance which allows the REPLACEMENT SHEET (RULE 26) interaction to take place. The interaction is eliminated expediently by hybridization with a complementary strand which is complementary to the first primer or by a synthesis which takes place on the first primer. The above-described procedure further reduces the possibility of contamination.
In a further embodiment of the method, the second fluorophoric molecule can also be bound to a second primer. However, it is also possible to incorporate nucleotides provided with the second fluorophoric molecule, or a further nucleic acid sequence, into a synthesis strand. The second primer is in solution.
After amplification and denaturation, the first and the second primer are advantageously hybridized in such a manner that the interaction is generated. The distance between the first and the second fluorophoric molecule in the hybridized state is preferably 2 to 12 nucleotides. The above variant of the method is particularly sensitive.
The solid phase can comprise a polymer which is preferably electroconductive, for example a polycarbonate, polycarbene, trimethylthiopene and/or triaminobenzene and/or carbon fibers. It has proved to be especially advantageous for the solid phase to be a microtiter plate.
In a further feature of an embodiment, the first molecule is an acceptor group and the second fluorophoric molecule a donor group. The acceptor group can be a 6-carboxytetramethylrhodamine and the donor group a 6-carboxyfluorescein. Other suitable donor/acceptor pairs can be seen from the table which follows:
REPLACEMENT SHEET (RULE 26) Donor Acceptor Fluorescein Fluorescein Fluorescein Tetramethylrhodamine IAEDANS (= 5-((((2- Fluorescein iodacyl) amino) ethyl) amino) -naphathalene-lsulon [sic]
acid) EDANS (=5-((2-aminomethyl)- DABCYL [sic] (4-dimethyl-amino)naphthalene-1- aminoazo-benzene-4'-sulfonic acid) sulfoylo chloride) [sic]
BODOPY [sic] FL BODIPY FL
Naturally, it is possible to swap the first and the second fluorophoric molecule. In a further feature of an embodiment, the first or second fluorophoric molecule can be replaced by a quencher, preferably a quencher formed by 4-[4'-dimethylaminophenylazo]benzoic acid.
Suitable quencher/fluorophore pairs can be seen from the table which follows:
Quencher Fluorophore DABCYL [sic] Coumarin DABCYL [sic] EDANS
DABCYL [sic] Fluorescein DABCYL [sic] Lucifer Yellow DABCYL [sic] Bodipy DABCYL [sic] Eosin DABCYL [sic] Tetramethylrhodamine DABCYL [sic] Texas Red DABCYL [sic] Erythrosin To determine the concentration of the nucleotide sequence to be detected, the fluorescence can be recorded by means of a fluorometer connected to a data REPLACEMENT SHEET (RULE 26) processing system, the concentration of the nucleotide sequence to be detected being determined from the change of the fluorescence intensity over time. The reference point used is preferably the second derivative of the fluorescence intensity over the number of the amplification cycles carried out.
In accordance with the solution with regard to the device, a microtiter plate is provided for carrying out the method according to the invention with an upper face which is provided with a plurality of well-shaped recesses and to which the first molecule is bound. A
first primer may be bound to the upper face, the first molecule advantageously being bound to the surface via the first primer. In a further embodiment, the first primer has a hairpin loop, and the first molecule is bound to one loop section and the second molecule opposite to a second loop section at a distance which allows the interaction to take place.
In a further embodiment with regard to the device, a kit is provided with a microtiter plate according to the invention and with a primer provided with a second molecule.
The method according to the invention is illustrated in greater detail with reference to the drawing. In this drawing, Fig. 1 shows the pairing of the strand and the complementary strand of a target DNA with a first and a second primer, Fig. 2 the hybridization of the primers synthesized, Fig. 3 the excitation of the fluorophoric molecules, REPLACEMENT SHEET (RULE 26) Fig. 4 the pairing of the strand and the complementary strand of a target DNA in a further variant of the method, Fig. 5 the excitation of the fluorophoric molecules in accordance with the variant of the method in Fig. 4, Fig. 6 the fluorescence of a detector nucleotide with and without nucleotide label, Fig. 7a a particle bound to a second primer following PCR with template DNA in a dark field image, Fig. 7b the particle of Fig. 7a in a fluorescence micrograph, Fig. 7c a particle bound to a second primer following PCR without template DNA in a dark field image, and Fig. 7d the particle of Fig. 7c in a fluorescence micrograph.
In Fig. l, a first primer Pl is bound to the upper face within a cavity of a microtiter plate M made of polycarbonate or polypropylene. The microtiter plate M
may contain a controlled resistance heating. It may also be an element of a resistance heating itself. A
first fluorophoric molecule Fl is bound to the first primer P1.
The nucleic acid sequence N to be detected, which is present in a target DNA, and the further components required for carrying out a polymerase chain reaction (PCR) or ligase chain reaction (LCR) are pipetted into the cavities. The latter comprise in particular a REPLACEMENT SHEET (RULE 26) _ 7 _ second primer P2 with a second fluorophoric molecule F2 bound thereto. The target DNA is denatured, i.e.
separated into a strand S and a complementary strand C.
The temperature is then reduced to 50 to 60° [sic]. The strand S binds with a complementary sequence segment to the first primer P1. The complementary strand G binds to the second primer P2 which is present in the fluid.
Then, the sequence segment which is missing in each case is synthesized by means of a Taq DNA polymerase.
The temperature is then raised to ~94°C, so that the synthesis strands comprising the fluorophoric molecules Fl, F2 are present in the fluid as single strands viz as synthesis strand SSl and as synthesis complementary strand SC1. The second fluorophoric molecule F2 may also be incorporated into the synthesis strand SSl in a form in which it is bound to nucleotides or a further nucleic acid sequence, instead of via the second primer P2. The temperature is reduced to 50 to 60° [sic] . The synthesis strand SS1 and the synthesis complementary strand SC1 hybridize, so that the first F1 and the second fluorophoric molecule F2 are present at a distance of 6 to 12 nucleotides. This is shown schematically in Fig. 2.
Upon excitation of the first fluorophoric molecule F1, which is designed as the donor, a radiation-free energy transfer to the second fluorophoric molecule F2, which acts as the acceptor, takes place. As a consequence, an increased fluorescence is observed on the second fluorophoric molecule F2. The fluorescence is detected by means of a fluorometer. The readings are transmitted to a data-processing system.
The first primer Pl may also exhibit a hairpin loop, the first fluorophoric molecule F1 being bound to a first loop section and a quencher being bound to a loop REPLACEMENT SHEET (RULE 26) _ g _ section opposite at a distance which allows the interaction to take place. When the hairpin loop is closed, the interaction causes the fluorescence to be quenched. As a result of hybridization with a complementary strand C which is complementary to the first primer P1 or by a synthesis which takes place on the first primer P1, the hairpin loop is opened up. The interaction between the fluorophoric molecule and the quencher is eliminated. Excitation of the fluorophoric molecules results in fluorescence.
Then, the next PCR cycle is started by raising the temperature. The synthesis strand SSl and the synthesis complementary strand SC1 are multiplied further, and, as a result, the fluorescence intensity is increased.
The change in fluorescence intensity over the number of PCR or LCR cycles is a measure for the initial concentration of the target DNA: the more target DNA a sample contains, the more rapidly the fluorescence intensity increases.
To carry out the abovementioned method, a microtiter plate M made of polycarbonate or polypropylene is used.
The first primer P1 is bound with its 5'-terminus in the well area to the upper face of the microtiter plate M to a polypropylene surface via a linker which is preferably composed of 6 CH2 groups. The first primer Pl is bound to the polypropylene surface by the method of Weiler-J. and Hoheisel-JD. (Anal.- [sic] Biochem., 1996; 243 (2) . 218-27).
Fig. 4 shows a further variant of the method. In this variant, the first fluorophoric molecule F1 is bound directly to the solid phase, i.e. the upper face of the microtiter plate M. The first primer P1 is bound to the solid phase in the vicinity of the first fluorophoric REPLACEMENT SHEET (RULE 26) molecule Fl. After hybridization of the synthesis strands SSl or the synthesis complementary strands SCl, excitation results in a radiation-free energy transfer from the first fluorophoric molecule F1 (donor) to the second fluorophoric molecule F2 (acceptor), where fluorescence results (Fig. 5).
Fig. 6 shows the fluorescence of PCR products of the PCR with primers which have 3'-fluorophores attached to them. The fluorescence of the PCR product has been measured in relative fluorescence units (RFU) at an excitation wavelength of 496 nm and an emission wavelength of 576 nm. The sample ~~PCR without template"
is a PCR preparation without HGH template DNA after 25 cycles . The sample '~PCR with template" is a PCR mix with HGH template DNA after 25 cycles. The column on the right shows the PCR mix with template DNA, but without temperature cycles having been carried out.
It can be seen clearly from Fig. 6 that the template can be detected readily with the aid of the method according to the invention, in particular without any need for washing steps.
Example l:
Fluorescence energy transfer in PCR products of primers which have 3'-fluorophores attached to them Two primers labeled with fluorophoric groups in the 3'-terminus zone are synthesized.
A first primer with a length of 23 bases has the following sequence:
5'-ACCAGGAGTTTGTAAGCTCTTGG-3'.
REPLACEMENT SHEET (RULE 26) wo 99/47700 PCT/DE99/00725 The thymidine in position 4 relative to the 3'-terminus (emboldened in the sequence) is labeled with 6-carboxyfluorescein (6-FAM). The FAM group is bound via the amino group of the dT-C2-NH2, which is incorporated during oligonucleotide synthesis.
A second primer with a length of 19 bases has the following sequence:
5'-biotin-CCTGATGCGCACCCATTCC-3' The thymidine in position 3 relative to the 3'-terminus (emboldened in the sequence) is labeled with carboxymehtylrhodamine [sic] (TAMRA). The TAMRA group is bound via the amino group of the dT-C2-NH2, which is incorporated during oligonucleotide synthesis. The second primer is labeled at the 5'-terminus with a biotin group.
The synthesis is carried out on a 0.2~mol scale. The primers are purified by HPLC. The sequences of the primers are in immediate vicinity of a sequence segment of the human growth hormone gene (HGH gene).
First primer: 5'-ACCAGGAGTTTGTAAGCTCTTGG-3' 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 which covers the sequence segment of the HGH gene. The PCR is carried out in a total volume of 501 with in each case 0.5~M primer, 2 units of Taq-DNA
polymerase and 1~1 of HGH gene (long) in the relevant PCR buffers (all solutions and enzymes from Boehringer, REPLACEMENT SHEET (RULE 26) 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.
As negative control, the same PCRs are carried out, but without the template DNA. As a further control, the PCR
mix is left at 4°C.
The PCR results in the formation of the PCR product in which the fluorophores of the first and second primers are arranged on the strands of opposite polarity at a distance of a few bases:
FAM
5'-ACCAGGAGTTTGTAAGCTCTTGG-GGAATGGGTGCGCATCAGG-3' 3'-TGGTCCTCAAACATTCGAGAACC-CCTTACCCACGCGTAGTCC-biotin-5' TAMRA
A suitable excitation of the 5-FAM group at 496nm results in a fluorescence energy transfer to the TAMRA
group which is located on the complementary strand and which has an emission maximum at 576nm.
To detect the formation of the PCR product and the fluorescence energy transfer, the fluorescence is determined in a fluorescence spectrometer at an excitation of 496nm(+/- lOnm) and an emission of 576 nm (+/- lOnm). Owing to the PCR, the fluorescence of the TAMRA group increases (Fig. 6). This increase in fluorescence shows the formation of the expected PCR
product.
REPLACEMENT SHEET (RULE 26) Example 2:
PCR with 3'-labeled and immobilized primers The primers which described in Example 1 are also used for the PCR with 3'-labeled and immobilized primers.
The 5'-biotinylated second primer in accordance with Example 1 is bound by the PCR to streptavidin-coated, superparamagnetic particles with a size of approx.
2.8 ~m in diameter (M-280 Dynabeads, Dynal, Hamburg). To this end, the particles (10~g/~1; 6.7 x 108 particles/ml suspended in phosphate-buffered saline (PBS) pH 7.4 are washed with B/W buffer (lOmM Tris-C1, 1mM EDTA, 2M
NaCl) ph [sic] 7.5 and brought to a concentration of 5 ~g/~1 in B/W buffer. 20~t1 of this suspension are treated with an equal volume of 50~M solution of primer-2 in distilled water. The suspension is incubated for 1 hour at room temperature with gentle shaking. Unbound primer is removed by washing the particles twice, first with 1001 of B/W buffer and then by washing with lOmM TrisCl, 0.2 mM EDTA pH8 (TE).
The particles are stored at 4°C in TE in a suspension of 10~g/~1.
The PCR with the second primer which is bound to the supermagnetic particle is carried out as in Example 1.
In contrast to Example 1, l~l of the suspension of the particle-bound primer-2 is employed instead of the free second primer. After the PCR, the particles are washed repeatedly with TE and analyzed on the fluorescence microscope. What is studied is the attachment of the 6-FAM-labeled first primer to the particles. Fig. 7A
demonstrates the fluorescence of the particles after conclusion. The PCR mix shown in Fig. 7B shows a fluorescence of the particles caused by the attachment of the FAM-labeled first primer to the particles. This fluorescence is not present in the control without template DNA (Fig. 7D).
REPLACEMENT SHEET (RULE 26) The symbols denote:
S Strand C Complementary strand P1 First primer P2 Second primer Fl First fluorophoric molecule F2 Second fluorophoric molecule SSl Synthesis strand SCl Synthesis complementary strand M Microtiter plate N Nucleotide sequence REPLACEMENT SHEET (RULE 26) SEQUENCE PROTOCOLS
<110> november AG Novus Medicatus Bertling Gesellschaft fur Molekolare [sic] Medizin <120> Method and device for detecting a nucleotide sequence <130> 390687ga5 <140>
<141>
<160> 4 <170> PatentIn Ver. 2.1 <210> 1 <211> 23 <212> DNA
<213> human <400> 1 accaggagtt tgtaagctct tgg 23 <210> 2 <211> 42 <212> DNA
<213> human <400> 2 accaggagtt tgtaagctct tggggaatgg gtgcgcatca gg 42 <210> 3 <211> 42 <212> DNA
<213> human REPLACEMENT SHEET (RULE 26) <400> 3 cctgatgcgc acccattccc caagagctta caaactcctg gt 42 <210> 4 <211> 19 <212> DNA
<213> human <400> 4 cctgatgcgc acccattcc 19 REPLACEMENT SHEET (RULE 26)
Claims (28)
1. Method for detecting a nucleotide sequence (N) by means of fluorescence, where, if the nucleotide sequence to be detected (N) is present, an interaction which allows a radiation-free energy transfer between a first (F1) and a second fluorophoric molecule (F2) is generated or eliminated, characterized in that a first primer (P1) labeled in the region of its 3'-terminus with the first fluorophoric molecule (F1) is bound to a solid phase (M), and where a complementary synthesis strand (SC1) formed using the first primer (P1) and a synthesis strand (SS1) formed using the second primer (P2) are hybridized in such a manner that the interaction is generated or eliminated.
2. Method according to Claim 1, where the first primer (P1) has a hairpin loop and the first fluorophoric molecule (F1) is bound to one loop section and the second molecule opposite to a second loop section at a distance which allows the interaction to take place.
3. Method according to one of the preceding claims, where the second fluorophoric molecule (F2) is bound to the second primer (P2).
4. Method according to one of the preceding claims, where nucleotides provided with the second fluorophoric molecule (F2) are incorporated into a synthesis strand (SS1).
5. Method according to one of the preceding claims, where the distance between the first (F1) and the second fluorophoric molecule (F2) in the hybridized state is 2 to 12 nucleotides.
6. Method according to one of the preceding claims, where the solid phase (M) comprises a polymer, preferably an electroconductive polymer.
7. Method according to Claim 6, where the polymer comprises a polycarbonate, trimethylthiophene, triaminobenzene and/or a polycarbene, and/or carbon fibers.
8. Method according to one of the preceding claims, where the solid phase (M) is a microtiter plate.
9. Method according to one of the preceding claims, where the fist (F1) or second fluorophoric molecule (F2) is replaced by a quencher, preferably a quencher formed by 4-[4'dimethylaminophenylazo]benzoic acid.
10. Method according to one of the preceding claims, where the first fluorophoric molecule (F1) is a donor group and the second fluorophoric molecule (F2) is a corresponding acceptor group.
11. Method according to Claim 10, where the acceptor group is 6-carboxytetramethylrhodamine, tetramethylrhodamine, fluorescein, DABCYL or Bodipy F1.
12. Method according to Claim 10 or 11, where the donor group is 6-carboxyfluorescein, fluorescein, IADEANS [sic], EDANS or Bodipy Fl.
13. Method according to one of the preceding claims, where the fluorescence is recorded by means of a fluorometer connected to a data processing system.
14. Method according to one of the preceding claims, where the concentration of the nucleotide sequence to be detected (N) is determined from the change in fluorescence intensity over time.
15. Microtiter plate for carrying out the method according to one of Claims 1 - 14 with an upper face provided with a plurality of well-shaped recesses, characterized in that a first primer (P1) which is labeled in the region of its 3'-terminus with a first fluorophoric molecule (F1) and which is suitable for forming a synthesis complementary strand (SC1) is bound to the upper face.
16. Microtiter plate according to Claim 15, where the first primer (P1) has a hairpin loop and the first fluorophoric molecule (F1) is bound to the one loop section and a second fluorophoric molecule (F2) opposite to the second loop section at a distance which allows the interaction to take place.
17. Microtiter plate according to Claim 16, where the first fluorophoric molecule (F1) is an acceptor group and the second fluorophoric molecule (F2) is a donor group.
18. Microtiter plate according to one of Claim 16 or 17, where the first fluorophoric (F1) or the second fluorophoric molecule (F2) is replaced by a quencher, preferably a quencher formed by 4-[4'-dimethylaminophenylazo]benzoic acid.
19. Microtiter plate according to Claim 17, where the acceptor group is 6-carboxytetramethylrhodamine, tetramethylrhodamine, fluorescein, DABCYL or Bodipy Fl.
20. Microtiter plate according to one the Claim [sic]
17 or 19 where the donor group is 6-carboxyfluorescein, fluorescein, IADEANS [sic], EDANS or Bodipy Fl.
17 or 19 where the donor group is 6-carboxyfluorescein, fluorescein, IADEANS [sic], EDANS or Bodipy Fl.
21. Microtiter plate according to one of Claims 15 to 20, where the microtiter plate (M) comprises a polymer, preferably an electroconductive polymer.
22. Microtiter plate according to Claim 21, where the polymer comprises a polycarbonate, trimethylthiophene, triaminobenzene and/or polycarbene and/or carbon fibers.
23. Kit with a microtiter plate according to one of Claims 15 to 22 and a second primer (P2) which is provided with a second fluorophoric molecule (F2).
24. Kit according to Claim 23, where the first fluorophoric molecule (Fl) is an acceptor group and the second fluorophoric molecule (F2) is a donor group.
25. Kit according to Claim 24, where the first (Fl) or and [sic] the second fluorophoric molecule (F2) is replaced by a quencher, preferably a quencher formed by 4-[4'-dimethylaminophenylazo]benzoic acid.
26. Kit according to Claim 24, where the acceptor group is 6-carboxytetramethylrhodamine, tetramethylrhodamine, fluorescein, DABCYL or Bodipy Fl.
27. Kit according to Claim 24 or 26, where the donor group is 6-carboxyfluorescein, fluorescein, IADEANS [sic], EDANS or Bodipy Fl.
28. Kit according to one of Claims 23 to 27 encompassing deoxynucleotide triphosphates, buffer components and enzymes required for amplification by means of PCR or LCR.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19811729A DE19811729C2 (en) | 1998-03-18 | 1998-03-18 | Method and device for detecting a nucleotide sequence |
| DE19811729.9 | 1998-03-18 | ||
| PCT/DE1999/000725 WO1999047700A1 (en) | 1998-03-18 | 1999-03-16 | Method and device for detecting a nucleotide sequence |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2323075A1 true CA2323075A1 (en) | 1999-09-23 |
Family
ID=7861305
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002323075A Abandoned CA2323075A1 (en) | 1998-03-18 | 1999-03-16 | Method and device for detecting a nucleotide sequence |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP1064407A1 (en) |
| JP (1) | JP2002506654A (en) |
| CA (1) | CA2323075A1 (en) |
| DE (1) | DE19811729C2 (en) |
| WO (1) | WO1999047700A1 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8247179B2 (en) | 2002-03-12 | 2012-08-21 | Enzo Life Sciences, Inc. | Processes for quantitative or qualitative detection of single-stranded nucleic acids |
| US9353405B2 (en) | 2002-03-12 | 2016-05-31 | Enzo Life Sciences, Inc. | Optimized real time nucleic acid detection processes |
| EP3118333A3 (en) * | 2006-12-13 | 2017-04-26 | Luminex Corporation | Systems and methods for multiplex analysis of pcr in real time |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE19960076C2 (en) * | 1999-12-13 | 2002-12-05 | November Ag Molekulare Medizin | Method and device for the detection and quantification of biomolecules |
| DE10111420A1 (en) * | 2001-03-09 | 2002-09-12 | Gnothis Holding Sa Ecublens | To detect an analyte by fluorescence correlation spectroscopy, a set gap is established between the light focusing unit and the volume of the sample, and the sample carrier is thermally insulated from the light source |
| JP4710283B2 (en) * | 2004-09-08 | 2011-06-29 | 富士レビオ株式会社 | Nucleic acid sequence amplification and detection methods |
| US9416153B2 (en) | 2011-10-11 | 2016-08-16 | Enzo Life Sciences, Inc. | Fluorescent dyes |
| JP7035972B2 (en) * | 2018-11-09 | 2022-03-15 | 横河電機株式会社 | Device for measuring nucleic acid sequences |
Family Cites Families (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4996143A (en) * | 1985-12-23 | 1991-02-26 | Syngene, Inc. | Fluorescent stokes shift probes for polynucleotide hybridization |
| BR9206705A (en) * | 1991-11-01 | 1995-11-21 | Adelaide Children S Hospital | Solid phase amplification process |
| EP0601889A2 (en) * | 1992-12-10 | 1994-06-15 | Maine Medical Center Research Institute | Nucleic acid probes |
| US5925517A (en) * | 1993-11-12 | 1999-07-20 | The Public Health Research Institute Of The City Of New York, Inc. | Detectably labeled dual conformation oligonucleotide probes, assays and kits |
| US5538848A (en) * | 1994-11-16 | 1996-07-23 | Applied Biosystems Division, Perkin-Elmer Corp. | Method for detecting nucleic acid amplification using self-quenching fluorescence probe |
| US5654419A (en) * | 1994-02-01 | 1997-08-05 | The Regents Of The University Of California | Fluorescent labels and their use in separations |
| US5945283A (en) * | 1995-12-18 | 1999-08-31 | Washington University | Methods and kits for nucleic acid analysis using fluorescence resonance energy transfer |
| WO1997031256A2 (en) * | 1996-02-09 | 1997-08-28 | Cornell Research Foundation, Inc. | Detection of nucleic acid sequence differences using the ligase detection reaction with addressable arrays |
| CA2658290C (en) * | 1996-06-04 | 2012-04-10 | University Of Utah Research Foundation | Monitoring hybridization during pcr using fluorescent dye specific to double-stranded dna |
-
1998
- 1998-03-18 DE DE19811729A patent/DE19811729C2/en not_active Expired - Fee Related
-
1999
- 1999-03-16 JP JP2000536883A patent/JP2002506654A/en not_active Withdrawn
- 1999-03-16 WO PCT/DE1999/000725 patent/WO1999047700A1/en not_active Application Discontinuation
- 1999-03-16 CA CA002323075A patent/CA2323075A1/en not_active Abandoned
- 1999-03-16 EP EP99919084A patent/EP1064407A1/en not_active Withdrawn
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8247179B2 (en) | 2002-03-12 | 2012-08-21 | Enzo Life Sciences, Inc. | Processes for quantitative or qualitative detection of single-stranded nucleic acids |
| US9068948B2 (en) | 2002-03-12 | 2015-06-30 | Enzo Life Sciences, Inc. | Processes for detection of nucleic acids |
| US9261460B2 (en) | 2002-03-12 | 2016-02-16 | Enzo Life Sciences, Inc. | Real-time nucleic acid detection processes and compositions |
| US9353405B2 (en) | 2002-03-12 | 2016-05-31 | Enzo Life Sciences, Inc. | Optimized real time nucleic acid detection processes |
| US10144957B2 (en) | 2002-03-12 | 2018-12-04 | Enzo Life Sciences, Inc. | Optimized real time nucleic acid detection processes |
| EP3118333A3 (en) * | 2006-12-13 | 2017-04-26 | Luminex Corporation | Systems and methods for multiplex analysis of pcr in real time |
| US10253354B2 (en) | 2006-12-13 | 2019-04-09 | Luminex Corporation | Systems and methods for multiplex analysis of PCR in real time |
| US11001877B2 (en) | 2006-12-13 | 2021-05-11 | Luminex Corporation | Systems and methods for multiplex analysis of PCR in real time |
Also Published As
| Publication number | Publication date |
|---|---|
| DE19811729A1 (en) | 1999-09-23 |
| WO1999047700A1 (en) | 1999-09-23 |
| EP1064407A1 (en) | 2001-01-03 |
| JP2002506654A (en) | 2002-03-05 |
| DE19811729C2 (en) | 2000-05-18 |
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