WO1999047700A1

WO1999047700A1 - Method and device for detecting a nucleotide sequence

Info

Publication number: WO1999047700A1
Application number: PCT/DE1999/000725
Authority: WO
Inventors: Wolf Bertling
Original assignee: november Aktiengesellschaft Gesellschaft für Molekulare Medizin
Priority date: 1998-03-18
Filing date: 1999-03-16
Publication date: 1999-09-23
Also published as: EP1064407A1; JP2002506654A; DE19811729A1; DE19811729C2; CA2323075A1

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

Method and device for detecting a nucleotide sequence

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.

Methods are known from US Pat. No. 4,996,143 and DE 195 81 489 T1 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 second primers are each provided with a fluorophore molecule. In the bonded state, the Förster effect leads to a non-radiative transfer of energy from one fluorophoric molecule to the other. This causes a specific fluorescence. - The known method is not particularly sensitive.

From US 5,607,834 it is known to use a primer with a hairpin loop to detect a nucleotide sequence. A fluorophoric molecule and a quencher are provided opposite each other on the loop sections of the hairpin loop. The distance between the fluorophoric molecule and the quencher enables a radiation-free energy transfer which quenches the fluorescence. However, if the primer hybridizes with a complementary counter strand, the hairpin loop is opened. The fluorescent quenching spatial relationship between the fluorophore molecule and the quencher is changed. Fluorescence can thus be observed.

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. In particular, a simple and inexpensive method with improved sensitivity and less contamination probability is to be specified. Furthermore, the concentration of the nucleotide sequence to be detected should be determinable as efficiently as possible.

This object is solved by the features of claims 1 and 19. Appropriate configurations result from the features of claims 2 to 18 and 20 to 34.

According to the invention, 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. By binding the at least one fluorophoric molecule to a solid phase, 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. According to a special embodiment, 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.

According to 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 second primers are advantageously hybridized in such a way that the interaction is generated. In the hybridized state, the distance between the first and second fluorophoric molecules is preferably 2 to 12 nucleotides. The aforementioned method variant is particularly sensitive.

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. According to a further design feature, 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:

Donor acceptor

Fluorescein fluorescein

Fluorescein tetramethylrhodamine

IAEDANS (= 5 - ((((2-fluorescein iodoacyl) amino) ethyl) amino) nap hathalene-isulonic acid)

EDANS (= 5 - ((2- DABCYL (4-dimethylaminoazo-aminomethyl) amino) naphthalene-benzene-4'-sulfoyl chloride) 1-sulfonic acid)

BODOPY FL BODIPY FL

It is of course possible for the first and second fluorophoric molecules to be interchanged. According to a further design feature, the first or second fluorophoric molecule can be replaced by a quencher, preferably formed from 4- [4 '-dimethylaminophenylazo] benzene acid.

Suitable quencher / fluorophore pairs are shown in the following table:

Quencher fluorophore

DABCYL Coumarin

DABCYL EDANS

DABCYL fluorescein

DABCYL Lucifer-Yellow

DABCYL Bodipy

DABCYL eosin

DABCYL tetramethylrhodamine

DABCYL Texas Red

DABCYL erythrosin

To determine the concentration of the nucleotide sequence to be detected, 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.

According to the solution on the device side, 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. According to a further design feature, 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. According to a further configuration on the device side, a kit with a microtiter plate according to the invention and a primer provided with a second molecule is provided.

The method according to the invention is explained in more detail with reference to the drawing. Show here

1 shows the pairing of the strand and the counter strand of a target DNA with a first and a second primer,

2 shows the hybridization of the synthesized primers,

3 the excitation of the fluorophoric molecules,

4 the pairing of the strand and the counter strand of a target DNA in a further method variant,

5 the excitation of the fluorophoric molecules according to the process variant in FIG. 4,

6 shows the fluorescence of a detector nucleotide with and without a labeling nucleotide,

7a shows a particle bound to a second primer after the PCR with template DNA in a dark field image,

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 and

7d the particle according to FIG. 7c in a fluorescence microscope image.

In Fig. 1, 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. 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

Liquid. Instead of via the second primer P2, 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.

When the first fluorophoric molecule F1, which is designed as a donor, is excited, there is a radiation-free energy transfer to the second fluorophoric molecule F2, which acts as an acceptor. As a result, 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. When 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. At the top of the microtiter plate M in the area of the wells, 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).

Another method variant is shown in FIG. 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).

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

and an emission wavelength of 576 nm were measured in relative fluorescence units (RFU). 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.

6 clearly shows that the template can be detected easily, in particular without the need for washing steps, with the aid of the method according to the invention.

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:

5'-ACCAGGAGTTTGTAAGCTCTTGG-3 '.

The thymidine at position 4 with respect to the 3 'end (printed in bold in the sequence) is labeled with 6-carboxyfluorescein (6-FAM). The FAM group is linked via the amino group of the dT-C2-NH2 incorporated during the oligonucleotide synthesis. 11

in the second primer with a length of 19 bases has the following sequence:

5 '-Biotin-CCTGATGCGCACCCATTCC-3'.

The thymidine at position 3 with respect to the 3 'end (printed in bold in the sequence) is marked with carboxymethylrhodamine (TAMRA). 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 μmol. 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).

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 covering the sequence portion of the HGH gene. The PCR is carried out in a total volume of 50 μl, each with 0.5 μM primer, 2 units of Taq DNA polymerase and 1 μl 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

As a negative control, the same PCR is carried out, leaving out the template DNA. As a further control, 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:

FAM

5 '-ACCAGGAGTTTGTAAGCTCTTGG-GGAATGGGTGCGCATCAGG-3' 3 '-TGGTCCTCAAACATTCGAGAACC-CCTTACCCACGCGTAGTCC-Biotin-5'

TAMRA

With a corresponding excitation of the 5-FAM group at 496nm, there is a fluorescence energy transfer to the TAMRA group in the opposite strand, whose emission maximum is 576nm.

To demonstrate the formation of the PCR product and the fluorescence energy transfer, 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:

PCR with 3 '-labeled and immobilized primer

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 μm in diameter (M-280 Dynabeads, Dynal, Hamburg). For this purpose, the particles (10 μg / μl; 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 μg / μl in B / W buffer To 20 μl of this suspension, the same volume of a 50 μm solution of primer-2 in distilled water is added Unbound primer is removed by washing the particles twice with lOOμl B / W buffer and then with lOmM TrisCl, 0.2mM EDTA pH8 (TE) .The particles are in a lOμg / μl suspension at 4 ° C stored in TE.

The PCR according to Example 1 is carried out with the second primer bound to the supermagnetic particle. In contrast to example 1, 1 μl of the suspension of particle-bound primer-2 is used instead of the free second primer. After the PCR, 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

which comes about through the attachment of the FAM-labeled first primer to the particles. This fluorescence is not present in the control without template DNA (FIG. 7D).

15

Reference list

S strand G counter strand

Pl first primer

P2 second primer

Fl first fluorophoric molecule

F2 second fluorophoric molecule SSI synthesis strand

SGI synthesis counter strand

M microtiter plate

N nucleotide sequence

16 ^{* "}

SEQUENCE PROTOCOLS

<110> november AG Novus Medicatus Bertling Society for Molecular Medicine

<120> Method and device for the detection of 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 17 ^{* "}

<211> 42 <212> DNA <213> human

<400> 3 cctgatgcgc acccattccc caagagctta caaactcctg gt 42

<210> 4 <211> 19 <212> DNA

<213> human

<400> 4 cctgatgcgc acccattcc 19

Claims

claims

1. A method for the detection of a nucleotide sequence (N) by means of fluorescence, an interaction between a first (Fl) and a second fluorophoric molecule (F2) being generated or eliminated when the nucleotide sequence (N) to be detected is present, which enables a radiation-free or direct energy transfer , characterized in that at least one of the fluorophoric molecules (Fl, F2) is bound to a solid phase (M).

2. The method according to claim 1, wherein a first primer (Pl) is bound to the solid phase (M).

3. The method according to any one of the preceding claims, wherein the first fluorophoric molecule (Fl) via the first primer (Pl) is bound to the solid phase (M).

4. The method according to claim 3, wherein the first primer (Pl) has a hairpin loop, and the first fluorophoric molecule (F1) are bound to one loop section and the second fluorophoric molecule (F2) opposite to the other loop section at an interaction-enabling distance.

A method according to claim 4, wherein the interaction is eliminated by hybridization with a complementary strand (G) complementary to the first primer (Pl) or by a synthesis taking place at the first primer (Pl).

Method according to one of the preceding claims, wherein the second fluorophoric molecule (F2) is bound to a second primer (P2). 19

7. The method according to any one of the preceding claims, wherein nucleotides provided with the second fluorophoric molecule (F2) or a further nucleic acid sequence are incorporated into a synthesis strand (SSI).

8. The method according to any one of the preceding claims, wherein the first (Pl) and the second primer (P2) are hybridized after amplification and denaturation so that the interaction is generated.

9. The method according to any one of the preceding claims, wherein in the hybridized state the distance between the first (F1) and second fluorophoric molecule (F2) is 2 to 12 nucleotides.

10. The method according to any one of the preceding claims, wherein the solid phase (M) contains a, preferably electrically conductive, plastic.

11. The method according to claim 10, wherein the plastic contains a polycarbonate, trimethylthiophene, triaminobenzene and / or a polycarbene, and / or carbon fibers.

12. The method according to any one of the preceding claims, wherein the solid phase is a microtiter plate (M).

13. The method according to any one of the preceding claims, wherein the first (F1) or second fluorophoric molecule (F2) is replaced by a quencher, preferably formed from 4- [4 'dimethylaminophenylazo] benzoic acid.

14. The method according to any one of the preceding claims, wherein the first fluorophore molecule (F1) is a donor group and the second fluorophore molecule (F2) is a corresponding acceptor group. 20th

15. The method according to any one of the preceding claims, wherein the acceptor group is 6-carboxy-tetramethyl-rhodamine, tetramethylrhodamine, fluorescein, DABCYL or Bodipy Fl.

16. The method according to any one of the preceding claims, wherein the donor group is 6-carboxy-fluorescein, fluorescein, IADEANS, EDANS or Bodipy Fl.

17. The method according to any one of the preceding claims, wherein the fluorescence is detected by means of a fluorometer connected to a data processing device.

18. The method according to any one of the preceding claims, wherein from the temporal change in the fluorescence intensity

Concentration of the nucleotide sequence to be detected (N) is determined.

19. Microtiter plate for carrying out the method according to one of claims 1-18, with a plurality of trough-shaped

Wells having top to which the first fluorophore molecule (Fl) is bound.

20. A microtiter plate according to claim 19, wherein a first primer (Pl) is bound to the top.

21. The microtiter plate according to claim 20, wherein the first fluorophoric molecule (F1) is bound to the top via the first primer (Pl).

22. A microtiter plate according to claim 21, wherein the first primer (Pl) has a hairpin loop, and the first fluorophoric molecule (F1) on one loop section and the second fluorophoric molecule (F2) opposite on the second- 21

th loop section are bound at a distance that enables the interaction.

23. Microtiter plate according to one of claims 19 to 22, wherein the first fluorophore molecule (F1) is an acceptor group and the second fluorophore molecule (F2) is a donor group.

24. Microtiter plate according to one of claims 19 to 22, wherein the first fluorophore (F1) or the second fluorophore molecule (F2) is replaced by a quencher, preferably formed from 4- [4'-dimethylaminophenylazo] benzoic acid.

25. A microtiter plate according to claim 23 or 24, wherein the acceptor group is 6-carboxy-tetramethyl-rhodamine, tetramethylrhodamine, fluorescein, DABCYL or Bodipy Fl.

26. The microtiter plate according to claim 25, wherein the donor group 6-carboxy-fluorescein, fluorescein, IADEANS,

EDANS or Bodipy Fl is.

27. Microtiter plate according to one of claims 19 to 26, wherein the microtiter plate (M) contains a, preferably electrically conductive, plastic.

28. A microtiter plate according to claim 27, wherein the plastic contains a polycarbonate, triethylthiophene, triaminobenzene and / or polycarbene and / or carbon fibers.

29. Kit with a microtiter plate according to one of claims 19 to 28 and a second primer (P2) provided with a second fluorophoric molecule (F2). 22

30. Kit according to claim 29, wherein the first fluorophore molecule (F1) is an acceptor group and the second fluorophore molecule (F2) is a donor group.

31. Kit according to claim 29, wherein the first (F1) or and the second fluorophoric molecule (F2) is replaced by a quencher, preferably formed from 4- [4 '-dimethylaminophenylazo] benzoic acid.

32. Kit according to claim 30 or 31, wherein the acceptor group is 6-carboxy-tetramethyl-rhodamine, tetramethylrhodamine, fluorescein, DABCYL or Bodipy Fl.

33. The kit of claim 32, wherein the donor group is 6-carboxy-fluorescein, fluorescein, IADEANS, EDANS or Bodipy Fl.

34. Kit according to one of claims 29 to 33, comprising deoxy nucleotide triphosphates, buffer components and enzymes required for amplification by means of PCR or LCR.