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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Citations (9)

• 1998
  • 1998-03-18 DE DE19811729A patent/DE19811729C2/de not_active Expired - Fee Related
• 1999
  • 1999-03-16 CA CA002323075A patent/CA2323075A1/en not_active Abandoned
  • 1999-03-16 WO PCT/DE1999/000725 patent/WO1999047700A1/de not_active Application Discontinuation
  • 1999-03-16 JP JP2000536883A patent/JP2002506654A/ja not_active Withdrawn
  • 1999-03-16 EP EP99919084A patent/EP1064407A1/de not_active Withdrawn

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