WO2003040680A2 - Fluoreszenz-quenchen zur detektion von nukleinsäure-oligomer-hybridisierungsereignissen bei hohen salz-konzentrationen - Google Patents
Fluoreszenz-quenchen zur detektion von nukleinsäure-oligomer-hybridisierungsereignissen bei hohen salz-konzentrationen Download PDFInfo
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- WO2003040680A2 WO2003040680A2 PCT/DE2002/004148 DE0204148W WO03040680A2 WO 2003040680 A2 WO2003040680 A2 WO 2003040680A2 DE 0204148 W DE0204148 W DE 0204148W WO 03040680 A2 WO03040680 A2 WO 03040680A2
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- 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 present invention relates to a method for the detection of nucleic acid-oligomer hybridization events by fluorescence quenching.
- Immunoassays and increasingly sequence analysis of DNA and RNA are used in disease diagnosis, in toxicological test procedures, in genetic research and development, as well as in the agricultural and pharmaceutical sectors.
- serial methods with autoradiographic or optical detection parallel detection methods using array technology, so-called DNA or protein chips, are increasingly being used. With these parallel methods, too, the detection is based on optical, radiographic, mass spectrometric or electrochemical methods.
- probe oligonucleotides For gene analysis on a chip, a library of known DNA sequences ("probe oligonucleotides”) is fixed in an ordered grid on one surface, so that the probe oligonucleotides.
- Target oligonucleotides present in the test solution, the sequences of which are complementary to certain probe oligonucleotides on the chip, can be identified by
- Radio label e.g. 32 P
- a fluorescent dye e.g. fluorescein, Cy3 TM or
- Fluorescence scanners are increasingly using radio labels.
- the fluorescence scanners currently available on the market enable the detection of
- Fluorophore in the subattomole range Fluorophore in the subattomole range.
- labeled targets to detect hybridization events has some drawbacks.
- the marking must take place before the actual measurement, which means an additional synthesis step and thus additional work. It is also difficult to ensure homogeneous marking of the sample material.
- stringent washing conditions are necessary in order to remove non-specific or non-specifically bound material following hybridization.
- targets antibodies or antigen or DNA fragment
- Probe molecules are labeled with appropriate fluorescent dyes.
- the so-called molecular beacons work according to this principle. These single-stranded oligonucleotides have a hairpin structure (stem-and-loop) and carry a fluorophore (e.g. Fluorescein, TexasRed®) at one end of the sequence and a suitable one at the other end of the sequence
- a fluorophore e.g. Fluorescein, TexasRed®
- Fluorescence quencher e.g. DABCYL Due to the special geometric arrangement, the fluorescent group and the unit that are used to extinguish the
- gold nanoparticles are also used as efficient quenchers (cf. Nature Biotech. Vol. 19, 2001, page 365).
- the quenching of fluorescence by metals is based primarily on a radiationless energy transfer from the dye molecule to the metal. A greater sensitivity is observed when using gold nanoparticles than with organic quenchers.
- dyes are efficiently quenched into the near infrared range.
- one disadvantage of this method is that gold nanoparticles are no longer stable at temperatures above 50 ° C. Another disadvantage is that this method is limited to the investigation of solutions and therefore only a few sequences can be examined at the same time, the degree of parallelization of this approach is therefore low.
- the object of the present invention is therefore to provide a method for the detection of nucleic acid-oligomer hybrids which does not have the disadvantages of the prior art.
- PNA Peptide nucleic acid synthetic DNA or RNA in which the
- N (COCH 2 base) -CH 2 CO unit hybridizes PNA with DNA.
- Nucleic acid at least two covalently linked nucleotides or at least two covalently linked pyrimidine (e.g. cytosine,
- Thymine or uracil or purine bases (e.g. adenine or
- nucleic acid refers to any one of nucleic acid.
- Bases such as on the sugar-phosphate backbone of the DNA, cDNA or RNA, on a peptide backbone of the PNA or on analogous structures (e.g. phosphoramide, thio-phosphate or
- Nucleic acid oligomer Nucleic acid of unspecified base length (e.g. Nucleic acid octamer: A nucleic acid with any backbone in which 8 pyrimidine or purine bases are covalently bound to each other). ns oligomer Nucleic acid oligomer
- Oligomer equivalent to nucleic acid oligomer Oligomer equivalent to nucleic acid oligomer.
- Oligonucleotide equivalent to oligomer or nucleic acid oligomer e.g. a DNA, PNA or RNA fragment of unspecified base length.
- Fluorophore chemical compound that is able to emit a longer-wave (red-shifted) fluorescent light when excited with light.
- Fluorophores fluorescent dyes
- UV ultraviolet
- VIS visible
- IR infrared
- the absorption and emission maxima are usually shifted by 15 to 40 nm (Stokes shift).
- Fluorescein resorcin phthalein fluorescent dye
- Rhodamine 6G Basic Red 1 fluorescent dye
- Texas Red® fluorescent dye from Molecular Probes, Inc.
- Quench surface conductive (metal) surface that can quench fluorescence through an energy transfer (especially gold, silver, copper surfaces etc.)
- linkers are commercially available as alkyl, alkenyl, alkynyl, hetero-alkyl, hetero-alkenyl or hetero-alkynyl chains, the chain being derivatized at two points with (identical or different) reactive groups. These groups form a covalent chemical bond in simple / known chemical reactions with the corresponding reaction partner.
- the reactive groups can also be photoactivatable, ie the reactive groups are only activated by light of certain or any wavelength.
- Preferred linkers are those of chain length 1-60, in particular chain length ⁇ -40, the chain length here being the shortest continuous connection between the structures to be connected, i.e. between the two molecules or between a surface atom, a surface molecule or a surface molecule group and another Molecule.
- Spacer linker which is covalently bonded via the reactive groups to one or both of the structures to be connected (see linker).
- Preferred spacers are those of chain length 1-60, in particular chain length ⁇ -40, the chain length being the shortest continuous connection between the structures to be connected.
- the terminal phosphate group of the oligonucleotide is esterified at the 3 'end with (HO- (CH 2 ) 2 -S) 2 to PO- (CH 2 ) 2 -SS- (CH 2 ) 2 -OH, the SS bond being cleaved homolytically and each causes an Au-SR bond.
- the probe oligonucleotide carries a covalently attached fluorophore (FP) such as Cy3 TM, Cy ⁇ TM, Texas Red®, Rhodamine 6G, fluorescein etc.
- FP covalently attached fluorophore
- Au-S- (CH 2 ) z-ds-oligo-Au-S-fCH ⁇ ⁇ ⁇ -ss-oligo-FP hybridizes with the oligonucleotide complementary to ss-oligo FP.
- the present invention relates to a method for detecting nucleic acid-oligomer hybridization events by fluorescence quenching, which comprises providing a modified surface as a first step.
- the modification of the surface consists in the attachment of at least one type of modified nucleic acid oligomer, the nucleic acid oligomers being modified by attachment of at least one type of fluorophore.
- the further steps of the method according to the invention are providing a sample with nucleic acid oligomers, bringing the sample into contact with the modified surface, setting a defined salt concentration of greater than 0.5 mol / l in the solution surrounding the modified nucleic acid oligomers, detection of the fluorescence of the fluorophore and comparison of the fluorescence intensity determined in the detection with reference values.
- a comparison of the detected fluorescence intensities with reference values is required in the method according to the invention. These reference values may already exist from previous measurements and therefore need most general case can not be detected in the course of the inventive method. However, since the reference values should ideally be determined under exactly the same external conditions as the actual measured values of the fluorescence intensities, according to a preferred embodiment of the present invention, a first detection of the fluorescence of the fluorophore is carried out before the targets (sample) come into contact with the modified surface , for this purpose, a defined salt concentration is set in the solution surrounding the modified nucleic acid oligomers, the same salt concentration being used as in the second detection of the fluorescence of the fluorophore, then the first detection of the fluorescence of the fluorophore takes place. Finally, stringency conditions for the hybridization are set and the sample is brought into contact with the modified surface. The values obtained in this first fluorescence detection are then used as reference values and compared with the values obtained in the second fluorescence detection.
- the stringency conditions for the hybridization and the contacting of the sample with the modified surface can in principle be carried out in any time sequence.
- the stringency conditions for the hybridization are preferably set simultaneously with the contacting of the sample with the modified surface, or the stringency conditions for the hybridization are set after the sample has been brought into contact with the modified surface.
- a normalization measurement is also carried out.
- sites are applied to the modified surface to which a very specific degree of association can be assigned after adding the sample.
- the signal obtained during the detection is then characteristic of this particular degree of association and can be used to normalize the signals from the test sites.
- the present invention namely also encompasses methods in which a modified surface is used which has been modified by binding at least two types of modified nucleic acid oligomers.
- the various types of modified nucleic acid oligomers are bound to the surface in essentially spatially separated areas. Under "essentially separate areas” are understood to mean areas of the surface that are predominantly modified by binding a certain type of modified nucleic acid oligomers. Only in areas in which two such essentially separated areas adjoin one another can spatial mixing of different types of modified nucleic acid oligomers occur.
- the nucleic acid oligomer is added to the sample before the sample is brought into contact with the modified surface, the nucleic acid oligomer being a binding partner with a high association constant with a particular type of modified nucleic acid oligomer which is bound to the surface in a certain area (Site T 10 o).
- the nucleic acid oligomer is added to the sample in an amount that is greater than the amount of nucleic acid oligomers that is necessary to fully associate the modified nucleic acid oligomers of the T 10 o sites.
- the last step of this method is the comparison of the values obtained in the detection of the fluorescence of the fluorophore with the value obtained for the region T 100 .
- the value obtained for the area T 10 o thus corresponds to the value when the association is complete (100%).
- a modified surface which has been modified by attaching at least three types of modified nucleic acid oligomers.
- the various types of modified nucleic acid oligomers are bound to the surface in essentially spatially separated areas.
- At least one type of modified nucleic acid oligomer is bound to the surface in a certain area (site T 0 ), which is known to contain no binding partner with a high association constant, i.e. the corresponding nucleic acid oligomer is not contained in the sample Sample occurs.
- a nucleic acid oligomer is added to the sample before the sample is brought into contact with the modified surface, the nucleic acid oligomer being a binding partner with a high association constant with a specific type of modified nucleic acid oligomer, which in a certain area (Site T 100 ) is bound to the surface.
- the nucleic acid oligomer is added to the sample in an amount that is greater than the amount of nucleic acid oligomers that is necessary to fully associate the modified nucleic acid oligomers of the T 10 o sites.
- the last step of this method is the comparison of the values obtained in the detection of the fluorescence of the fluorophore with that for the area T 100 obtained value and with the value obtained for the area T 0 .
- the value obtained for the area To corresponds to the value in the absence of association (0%).
- At least one further type of nucleic acid oligomer is added to the sample, it being known that this nucleic acid oligomer is not in the original sample is included.
- This further type of nucleic acid oligomer has an association constant> 0 to a type of modified nucleic acid oligomer which is present in a certain region (site T n ) bound to the surface.
- the nucleic acid oligomer is added to the sample in such an amount that after contacting the sample with the modified surface, n% of the modified nucleic acid oligomers of the site T n are in an associated form.
- the last step of this method is the comparison of the values obtained in the detection of the fluorescence of the ⁇ fluorophore with the value obtained for the area Tioo, with the value obtained for the area T 0 and with the values obtained for the areas T n .
- the value obtained for a specific test site T n thus corresponds to the value in the presence of n% associates from modified nucleic acid oligomers and target nucleic acid oligomers based on the total number of modified nucleic acid oligomers of 0 in accordance with Art.
- the amount of nucleic acid oligomers which has to be brought into contact with the modified surface in order to bring about an n% association at the site T n can be determined by the person skilled in the art by simple routine tests. For this purpose, for example after ⁇ detection of the values for T 0 and T 10 o, a calibrated measurement is carried out in which the signal intensity is determined by (different) detection labels with which the modified nucleic acid oligomers and the target nucleic acid oligomers are equipped. The ratio of the intensities of the target nucleic acid oligomer label signal to the modified nucleic acid oligomer label signal corresponds to n%. 0
- a normalization curve can be recorded with high accuracy. Standardizing the measurements of the actual test sites using this standardization curve significantly improves the reproducibility of the analysis using chip technology. ⁇ It should be pointed out that finding a nucleic acid oligomer that is not contained in the sample presents no problems, since even the largest genomes still offer a sufficient selection of sequences that are not available. In the event that the non-existing sequence differs from a ⁇ sequence present only by a base, the hybridization step must be carried out under stringent conditions. However, preference is given to using sequences which differ significantly, that is to say in several bases, from the sequences present in the sample. Particularly good results are achieved if oligonucleotides with the same or at least a similar number of bases are used for the test sites and for the standardization sites.
- the present invention also relates to a kit which comprises a modified surface, the modification consisting in the attachment of at least one type of modified nucleic acid oligomer and the nucleic acid oligomers being modified by ⁇ attachment of at least one type of fluorophore.
- the detection of the fluorescence is carried out after setting a defined salt concentration of greater than 0.1 mol / l in the solution surrounding the modified nucleic acid oligomers.
- Preferred salt concentrations are in the 0 ranges between 0, ⁇ and 10 mol / l, between 1 and 10 mol / l, between 0, ⁇ and 3 mol / l and in particular between 2 and 3 mol / l, because it was found in these ranges that there is a particularly large difference in the fluorescence intensity between the hybridized and the non-hybridized nucleic acid oligomer. This enables a particularly reliable detection of the hybridization events. ⁇
- surface refers to any carrier material that is suitable for carrying 0 or after corresponding chemical modification fluorophore-derivatized nucleic acid oligomers that are covalently immobilized on the surface and whose fluorescence is close to the surface (in approx. 1 to ⁇ O A distance to the surface) is significantly reduced by fluorescence quenching (radiation-free energy transfer between the fluorophore as emitter and the surface as absorber) (> 10% of the expected ⁇ fluorescence intensity of the fluorophore in the absence of the surface under otherwise identical conditions).
- fluorescence quenching radiation-free energy transfer between the fluorophore as emitter and the surface as absorber
- gold and silver are quenched Suitable surface material.
- the term surface is independent of the spatial dimensions of the surface and also includes nanoparticles (particles or clusters made up of a few individual to several hundred thousand surface atoms or molecules).
- the surface can also be bound to a solid support such as glass, metal 5 or plastic.
- Immobilization can e.g. covalently via amino, hydroxyl, epoxy or carboxy groups of the support material with thiol, hydroxyl, amino or carboxyl groups naturally present on the nucleic acid oligomer or by derivatization attached to the nucleic acid oligomer.
- the ⁇ nucleic acid oligomer can be connected directly or via a linker / spacer to the surface atoms or molecules of a surface.
- the nucleic acid oligomer can be anchored by the methods customary in immunoassays, e.g.
- nucleic acid oligomers for non-covalent immobilization on avidin or streptavidin-modified surfaces.
- the chemical modification of the probe nucleic acid oligomers with a surface anchor group can already be introduced in the course of automated solid phase synthesis or in separate reaction steps.
- the nucleic acid oligomer is also linked directly or via a linker / spacer to the surface atoms or molecules of a surface of the type described above. This binding can be carried out in various ways known from the prior art (see e.g. Hartwich, G .: ELECTROCHEMICAL DETECTION OF SEQUENCE-SPECIFIC
- nucleic acid oligomers When connecting the nucleic acid oligomers, care must be taken that they are either bound to the surface completely without additional co-adsorbate or, if a co-adsorbate appears necessary, that it forms a layer as thin as possible above the surface. Either a direct attachment of the nucleic acid oligomer to the surface must be carried out or it must be coated with short-chain co-adsorbates such as short-chain thiols. Co-adsorbates of chain length 1 to 30, preferably 1 to 20, particularly preferably 1 to 10, particularly preferably chain length 1 to 5 are preferred.
- a particular disadvantage in this connection is the binding of the nucleic acid oligomers in the form of a surface-biotin-avidin-biotin-oligomer compound.
- the fluorophore is always shielded from the surface by a very thick layer of biotin-avidin-biotin, which is associated with corresponding disadvantages in the detection of fluorescence.
- fluorescent dyes such as e.g. Texas Red®, rhodamine dyes, cyanine dyes such as Cy3 TM, Cy ⁇ TM, Fluorescein etc (see Fluka, Amersham and Molecular Probes catalog) are used.
- Fluorescence quenching is the deactivation of an electronically excited species via a radiationless process. Deactivation can take place by means of impacts or by radiation-free energy transfer to metals. The energy released is dissipated as thermal energy. Gold is an example of a metal that has the ability to quench fluorescence. The quenching has a strong dependence on the distance of the fluorophore from the surface functioning as a fluorescence quencher (inversely proportional to a higher (third to sixth) power of the distance). The effect of fluorescence quenching can therefore only be measured at distances of less than 100 to 200 ⁇ . In the range greater than approx. 200 A, further changes in distance no longer lead to a measurable increase in the fluorescence intensity. Brief description of the drawings
- 1 shows a schematic illustration of the detection of nucleic acid-oligomer hybridization events by modulation of the fluorescence quenching on quench surfaces
- oligomer probe 202 probe hybridizes with target 203: surface (e.g. gold) 204: distance from fluorophore to gold surface before hybridization 205: distance from fluorophore to gold surface after hybridization
- target 203 surface (e.g. gold)
- 204 distance from fluorophore to gold surface before hybridization
- 205 distance from fluorophore to gold surface after hybridization
- FIG. 1 shows a schematic illustration of the detection of nucleic acid-oligomer hybridization events by modulation of the fluorescence quenching on quench surfaces.
- FIG. 1A shows the case in which the single-stranded probe nucleic acid oligomer 201 is present in a form before the hybridization, which is characterized by a large distance 204 from the fluorophore 102 and the quenching metal surface.
- the distance 205 between the fluorescent dye molecule and the metal surface 203 functioning as a quencher. This leads to a reduction in the fluorescence intensity (bar chart in FIG. 1A).
- FIG. 1A shows the case in which the single-stranded probe nucleic acid oligomer 201 is present in a form before the hybridization, which is characterized by a large distance 204 from the fluorophore 102 and the quenching metal surface.
- the distance 205 between the fluorescent dye molecule and the metal surface 203 functioning as a quencher. This leads to a reduction in the
- 1B shows the case in which the single-stranded probe nucleic acid oligomer 201 is present in a form prior to hybridization, which is characterized by a small distance 204 from fluorophore 102 and quenching metal surface 203.
- the hybridization with the complementary nucleic acid oligomer strand 202 (target) increases the distance 20 ⁇ between the fluorescent dye molecule and the metal surface 203 functioning as quencher. This leads to an increase in the fluorescence intensity (bar chart 0 in FIG. 1B).
- FIG. 2A shows a plot of the fluorescence intensities of a 20-mer and a 30-mer nucleic acid probe (single-stranded) as a function of the ionic strength (here salt concentration) of the solution above the surface.
- the ⁇ fluorescence intensity of 200 ⁇ m spots of single-strand probe oligonucleotides (20 mer and 30 mer) immobilized on a 1 cm 2 gold plate was measured with Cy3 TM as the covalently bound fluorophore.
- FIG. 2B shows the fluorescence intensity before and after the sequence-specific hybridization of a 30-mer nucleic acid probe with the complementary counter strand (target) as a function of 0 the salt concentration of the solution above the surface under otherwise the same conditions as in connection with FIG. 2A described. It is clear from FIG. 2B that at salt concentrations of more than 0.1 mol / l the fluorescence intensity increases by a factor of 5 after the hybridization. This allows a clear detection of the 5 hybridizations that have taken place, even in parallel methods.
- nucleic acid-oligomer probes of different sequences are bound to a support using the immobilization techniques described above.
- the hybridization event of any target Nucleic acid oligomers or a (fragmented) target DNA can be detected, for example, to detect mutations in the target and to detect them in a sequence-specific manner.
- the surface atoms or molecules of a defined area are linked on a surface with DNA ⁇ / RNA / PNA nucleic acid oligomers of known but any sequence, as described above.
- the DNA chip can also be derivatized with a single probe oligonucleotide.
- Nucleic acid oligomers eg DNA, RNA or PNA fragments
- base length 3 to 70 preferably length 5 to 60, particularly preferably length 10 to ⁇ O, particularly preferably 0 length 12 to, are used as probe nucleic acid oligomers 40 used.
- the target oligonucleotides can also comprise a larger number of bases, ie they can be longer than the probe oligonucleotides.
- the expression “ ⁇ nucleic acid oligomer complementary to the probe oligonucleotide” is understood to mean a nucleic acid oligomer which has a base sequence which is complementary to the probe oligonucleotide in a partial region. The remaining portion (s) of the nucleic acid oligomer then protrude at the end (s) of the probe oligonucleotide beyond its base chain.
- a reference measurement e.g. with a fluorescence scanner
- the fluorescence intensity of the fluorophore-labeled probe oligonucleotides in the single-stranded state is determined at a defined, previously set salt concentration in the surrounding solution.
- the (as concentrated as possible) test solution with target oligonucleotide (s) is added to the surface with immobilized probe oligonucleotides under stringent conditions for the hybridization.
- Hybridization only occurs in the case in which the solution contains target nucleic acid oligomer strands which are 0 complementary to the probe-nucleic acid oligomers bound to the surface, or at least in many areas complementary.
- ⁇ fluorescence intensity in the hybridized, double-stranded state is determined in a second fluorescence measurement.
- the difference between the reference measurement and the second measurement for each test site is proportional to the number of complementary (or in many areas complementary) target oligonucleotides originally present in the test solution for the respective test site.
- the reference measurement can be omitted if the size of the reference signal is known beforehand (e.g. from previous measurements etc.).
- ⁇ Due to the hybridization of probe-nucleic acid oligomer and the complementary nucleic acid-oligomer strand (target), the distance between the fluorescent dye molecule and the metal surface acting as quencher changes. Due to the changed distance, the extent of the quench process and thus the intensity of the fluorescence also undergoes a major change. Thus ⁇ can be a sequence specific hybridization event by fluorescence-based methods such as Fluorescence microscopy or measurements with fluorescence scanners can be detected.
- the single-stranded probe nucleic acid oligomer 201 is in a form which is characterized by a large distance 204 from fluorophore 102 and ⁇ quenching metal surface 203 (high fluorescence intensity).
- the distance 20 ⁇ between the fluorescent dye molecule 102 and the metal surface 203 functioning as a quencher changes in such a way that the reduction in the distance leads to an increase in quenching and 0 a lower intensity of fluorescence can be observed after hybridization (see FIG. 1A).
- the single-stranded probe nucleic acid oligomer 201 is in a form which is characterized by a short distance 204 from fluorophore 102 and ⁇ quenching metal surface 203 (low fluorescence intensity).
- the distance 20 ⁇ between the fluorescent dye molecule 102 and the metal surface 203 functioning as a quencher changes in such a way that the increase in the distance leads to a reduction in the quenching and a higher intensity of the fluorescence can be observed after the hybridization ⁇ ( see Figure 1 B).
- the two geometric arrangements shown above can be achieved by varying the ionic strength, in particular the salt concentration. Any salts can be used, with the exception of bivalent salts (eg Mg 2+ ) or 0 chaotropic salts.
- bivalent salts eg Mg 2+
- 0 chaotropic salts eg Mg 2+
- the surface-bound single-stranded probe is in a rather elongated conformation (see Figure 1A).
- a decrease in the fluorescence intensity is observed as a result of the hybridization (see FIGS. 1A, 2B).
- the surface-bound single-stranded probe With a high salt content, the surface-bound single-stranded probe is in a rather compressed conformation (see FIG. 1B).
- FIGS. 1B, 2B As a result of ⁇ the hybridization, an increase in the fluorescence intensity is observed (see FIGS. 1B, 2B).
- n nucleotide (nt) long nucleic acid probe (DNA, RNA or PNA, for example a 20 nucleotide long oligo) is near one of its ends (3 'or ⁇ ' end) directly or via (any) spacer a reactive group for covalent anchoring to ⁇ of the surface, e.g. as a 3'-thiol-modified probe oligonucleotide, in which the terminal thiol modification serves as a reactive group for binding to gold.
- Other covalent anchoring options arise e.g. from amine-modified oligonucleotide, which is used for anchoring to surface-bonded carboxylic acid functions (e.g.
- a fluorophore is covalently bound in the vicinity of the other terminus of the probe oligonucleotide (cf. Example 1).
- the nucleic acid probe modified in this way is
- Probe nucleic acid oligomers attached to the surface which may be correspondingly derivatized.
- the surface modified in this way is then brought into contact with the corresponding monofunctional linker in solution (for example alkanethiols or ⁇ -hydroxyalkanethiols in phosphate buffer / EtOH mixtures 0 in the case of thiol-modified probe oligonucleotides), the monofunctional linker being attached via its reactive group which - if appropriate derivatized - binds the surface (see section "Binding of the nucleic acid oligomers to the surface").
- the (residual) fluorescence of the fluorophore on the probe oligonucleotide is detected by a suitable method, for example by fluorescence measurement with a fluorescence scanner in the presence of different salt concentrations (cf. Example 7).
- the fluorescence intensity of the single-stranded probe oligonucleotide shows a maximum at a salt concentration between 0.0 ⁇ and 0.2 ⁇ mol / l (see FIG. 2A).
- potential hybridization events are made possible under suitable conditions known to the person skilled in the art (arbitrary, freely selectable stringency conditions of the parameters potential / temperature / salt / chaotropic salts etc.
- the difference in the measurement signal is proportional to the number of hybridization events between probe nucleic acid oligomer on the surface and suitable target nucleic acid oligomer in the test solution (see example 8 ).
- the method described can be for a target type (for example a specific target oligonucleotide type with a known sequence) on a surface or - if different probe types are used for each test site - for several target types (several different target oligonucleotide types) be applied.
- a target type for example a specific target oligonucleotide type with a known sequence
- the synthesis of the oligonucleotides takes place in an automatic oligonucleotide synthesizer (Expedite 8909; ABI 384 DNA / RNA synthesizer) according to the synthesis protocols recommended by the manufacturer for a 1.0 ⁇ mol synthesis.
- the oxidation steps are carried out with a 0.02 M iodine solution in order to avoid oxidative cleavage of the disulfide bridge.
- Modifications to the 5 'position of the oligonucleotides are carried out with a coupling step that is extended to ⁇ min.
- the amino modifier C2 dT (Glen Research 10-1037) is built into the sequences with the respective standard protocol ⁇ .
- the coupling efficiencies are determined online during the synthesis via the DMT cation concentration photometrically or conductometrically.
- the oligonucleotides are deprotected with concentrated ammonia (30%) at 37 ° C for 16 h.
- the oligonucleotides are purified using RP-HPL chromatography 0 according to standard protocols (eluent: 0.1 M triethylammonium acetate buffer, acetonitrile), and the characterization is carried out using MALDI-TOF MS.
- the amine-modified oligonucleotides are coupled to the correspondingly activated fluorophores (for example fluorescein isothiocyanate) in accordance with conditions known to the person skilled in the art. The coupling can take place both before and after the oligonucleotides have been bound to the surface.
- fluorophores for example fluorescein isothiocyanate
- oligonucleotide synthesizer (Expedite 8909; ABI 384 DNA / RNA synthesizer) according to the synthesis protocols recommended by the manufacturer for a 1.0 ⁇ mol synthesis.
- the 0 oxidation steps are carried out with a 0.02 M iodine solution in order to avoid oxidative cleavage of the disulfide bridge.
- Modifications to the ⁇ 'position of the oligonucleotides are carried out with a coupling step that is extended to ⁇ min.
- the fluorophores are incorporated into the sequences as phosphoramidites (Glen Research 10-1037) in the sequences with the respective standard protocol.
- the ⁇ coupling efficiencies are determined photometrically or conductometrically online via the DMT cation concentration during the synthesis.
- the quench surface (here: gold plate) is treated with a double-modified 20 bp single-strand oligonucleotide of the sequence ⁇ '-AGC GGA TAA CAC AGT CAC CT-3 ' ⁇ (modification 1: the phosphate group of the 3' end is marked with (HO - (CH 2 ) 2 -S) 2 esterified to PO- (CH 2 ) 2 - SS- (CH 2 ) 2 -OH; modification 2: at the ⁇ 'end is the fluorescein modifier fluorescein phosphoramidite (Proligo Biochemie GmbH ) built in according to the respective standard protocol) in ⁇ x10 "5 molar buffer solution (phosphate buffer, 0, ⁇ molar in water, pH 7) with addition of approx.
- modification 1 the phosphate group of the 3' end is marked with (HO - (CH 2 ) 2 -S) 2 esterified to PO- (CH 2 ) 2 - SS- (CH 2 ) 2
- the disulfide spacer PO- (CH 2 ) 2 -SS- (CH 2 ) 2 -OH of the oligonucleotide is cleaved homolytically, thereby forming the spacer with Au atoms on the surface to form a covalent Au S bond, which leads to coadsorption of the ss-oligonucleotide and the cleaved 2-hydroxy-mercaptoethanol s Free propanethiol which is simultaneously present in the ⁇ incubation solution is also adsorbed by forming an Au-S bond (incubation step).
- this single strand can also be hybridized with its complementary strand.
- the quench surface (here: gold plate) is coated with a double-modified 20 bp ⁇ single-strand oligonucleotide of the sequence ⁇ '-AGC GGA TAA CAC AGT CAC CT-3 '
- this single strand ⁇ can also be hybridized with its complementary strand.
- the gold surface modified in this way is completely wetted with an approx. 10 "5 to 10 ⁇ 1 molar solution of short-chain alkanethiols such as propanethiol (in water or buffer, pH 7 - 7. ⁇ or in ethanol) and 0.5 -
- short-chain alkanethiols such as propanethiol (in water or buffer, pH 7 - 7. ⁇ or in ethanol) and 0.5 -
- the free thiol occupies the free gold surface remaining after the incubation step by forming an Au-S bond, or alternatively other functional thiols or disulfides of suitable chain length with the same or different functional groups can be used.
- the probe surface is produced analogously to Example 4.
- a modified oligonucleotide of the sequence ⁇ '-fluorescein-AGC GGA TAA CAC AGT CAC CT-3 '[C 3 -S- SC 3 -OH] is immobilized on gold ( ⁇ O ⁇ mol oligonucleotide in phosphate buffer (K 2 HPO 4 / KH 2 PO ⁇ OO mM, pH 7, subsequent coating with propanethiol 1 mM in water) and in 0 in the form Au-S (CH 2 ) 2 -ss-oligo-fluorescein the fluorescence intensity of the surface with a fluorescence scanner from avision Biotech in To measure the fluorescence in the presence of liquid media, add 1 ⁇ 0 ⁇ l of the medium to the gold surface and then cover it with a cover glass, or alternatively use hybrid wells or imaging ⁇ chambers.
- the probe surface is produced analogously to example 4 and measured analogously to example 5.
- the geometric arrangement of the single-stranded 5 probe is modulated by varying the salt concentration (NaCl concentration) in a range between 1x10 "4 and 3 mol / liter.
- the values obtained by measuring the fluorescence intensity as a function of the salt concentration are shown in FIG. 2A
- the fluorescence intensity is highest in the concentration range between 0.01 and 0.5 mol / liter, but especially in the range between 0.0 ⁇ and 0.2 ⁇ mol / liter, ie the fluorophore is at the greatest distance from the gold surface
- Fluorescence measurements performed with a fluorescence scanner After hybridization with complementary oligomers in phosphate buffer ( ⁇ OO mM, 1 mM EDTA, 1 M NaCI), fluorescence measurements are carried out according to Example 6 in the presence of NaCI solutions of 0 different concentrations using a fluorescence scanner. The values obtained by measuring the fluorescence intensity for the hybridized and the non-hybridized case as a function of the salt concentration are shown in FIG. 2B.
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Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP02802612A EP1567664A2 (de) | 2001-11-09 | 2002-11-08 | Fluoreszenz-quenchen zur detektion von nukleinsäure-oligomer-hybridisierungsereignissen bei hohen salz-konzentrationen |
US10/529,472 US20060188877A1 (en) | 2001-11-09 | 2002-11-08 | Fluorescence-quenching used to detect nucleic acid oligomer hybrization events at high salt concentrations |
AU2002363453A AU2002363453A1 (en) | 2001-11-09 | 2002-11-08 | Fluorescence-quenching used to detect nucleic acid oligomer hybridization events at high salt concentrations |
Applications Claiming Priority (2)
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DE10155055.3 | 2001-11-09 | ||
DE10155055A DE10155055A1 (de) | 2001-11-09 | 2001-11-09 | Fluoreszenz-Quenchen zur Detektion von Nukleinsäure-Oligomer-Hybridisierungsereignissen bei hohen Salz-Konzentrationen |
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WO2003040680A2 true WO2003040680A2 (de) | 2003-05-15 |
WO2003040680A3 WO2003040680A3 (de) | 2004-01-29 |
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PCT/DE2002/004148 WO2003040680A2 (de) | 2001-11-09 | 2002-11-08 | Fluoreszenz-quenchen zur detektion von nukleinsäure-oligomer-hybridisierungsereignissen bei hohen salz-konzentrationen |
Country Status (5)
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US (1) | US20060188877A1 (de) |
EP (1) | EP1567664A2 (de) |
AU (1) | AU2002363453A1 (de) |
DE (1) | DE10155055A1 (de) |
WO (1) | WO2003040680A2 (de) |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6040138A (en) * | 1995-09-15 | 2000-03-21 | Affymetrix, Inc. | Expression monitoring by hybridization to high density oligonucleotide arrays |
FR2805348A1 (fr) * | 2000-02-23 | 2001-08-24 | Commissariat Energie Atomique | Analyse de cibles biologiques utilisant une biopuce comportant un marqueur fluorescent |
US6312906B1 (en) * | 1999-01-15 | 2001-11-06 | Imperial College Innovations, Ltd. | Immobilized nucleic acid hybridization reagent and method |
WO2002018951A2 (en) * | 2000-08-29 | 2002-03-07 | The Rockefeller University | Methods employing fluorescence quenching by metal surfaces |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
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US5824473A (en) * | 1993-12-10 | 1998-10-20 | California Institute Of Technology | Nucleic acid mediated electron transfer |
DE10049527A1 (de) * | 2000-10-06 | 2001-09-06 | Friz Biochem Gmbh | Thermostabile,photoinduzierbar redoxaktive Einheit zur elektrochemischen Detektion von Nukleinsäure-Oligomer-Hybridisierungsereignissen |
-
2001
- 2001-11-09 DE DE10155055A patent/DE10155055A1/de not_active Withdrawn
-
2002
- 2002-11-08 AU AU2002363453A patent/AU2002363453A1/en not_active Abandoned
- 2002-11-08 WO PCT/DE2002/004148 patent/WO2003040680A2/de not_active Application Discontinuation
- 2002-11-08 US US10/529,472 patent/US20060188877A1/en not_active Abandoned
- 2002-11-08 EP EP02802612A patent/EP1567664A2/de not_active Withdrawn
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6040138A (en) * | 1995-09-15 | 2000-03-21 | Affymetrix, Inc. | Expression monitoring by hybridization to high density oligonucleotide arrays |
US6312906B1 (en) * | 1999-01-15 | 2001-11-06 | Imperial College Innovations, Ltd. | Immobilized nucleic acid hybridization reagent and method |
FR2805348A1 (fr) * | 2000-02-23 | 2001-08-24 | Commissariat Energie Atomique | Analyse de cibles biologiques utilisant une biopuce comportant un marqueur fluorescent |
WO2002018951A2 (en) * | 2000-08-29 | 2002-03-07 | The Rockefeller University | Methods employing fluorescence quenching by metal surfaces |
Non-Patent Citations (2)
Title |
---|
ANONYMOUS: "SSC Buffer Concentration Saline-Sodium Citrate (SSC) buffer for ISH Procedures" INTERNET ARTICLE , [Online] XP002248057 Gefunden im Internet: <URL:http://www.zymed.com/pdf/00-xxxx/00-8 400.pdf> * |
DUBERTRET B ET AL: "Single-mismatch detection using gold-quenched fluorescent oligonucleotides" NATURE BIOTECHNOLOGY, NATURE PUBLISHING, US, Bd. 19, Nr. 4, April 2001 (2001-04), Seiten 365-370, XP002224627 ISSN: 1087-0156 in der Anmeldung erw{hnt * |
Also Published As
Publication number | Publication date |
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EP1567664A2 (de) | 2005-08-31 |
AU2002363453A1 (en) | 2003-05-19 |
DE10155055A1 (de) | 2003-05-28 |
WO2003040680A3 (de) | 2004-01-29 |
US20060188877A1 (en) | 2006-08-24 |
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