WO2013091614A1

WO2013091614A1 - Method for the electrochemical detection of nucleic acid oligomer hybridisation occurrences

Info

Publication number: WO2013091614A1
Application number: PCT/DE2012/100295
Authority: WO
Inventors: Gerhard Hartwich
Original assignee: Friz Biochem Gesellschaft Für Bioanalytik Mbh
Priority date: 2011-12-19
Filing date: 2012-09-21
Publication date: 2013-06-27
Also published as: DE102011056606B3

Abstract

The invention relates to a method for the electrochemical detection of nucleic acid oligomer hybridisation occurrences, comprising the steps of: a) preparing a modified surface, wherein the modification consists of the bonding of at least one type of probe nucleic acid oligomers (1), wherein the probe nucleic acid oligomers (1) have a probe pin section (5) complementary to a first signal probe section (5') of signal nucleic acid oligomers (2); b) preparing at least one type of signal nucleic acid oligomers (2), wherein the signal nucleic acid oligomers have a first signal pin section (5') complementary to the probe pin section (5) of the probe nucleic acid oligomers (1), the signal nucleic acid oligomers (2) have a signal recognition section (8) complementary to a target recognition section (8') of target nucleic acid oligomers (10), the signal nucleic acid oligomers (2) have a first and a second signal pin section (5, 5'') complementary to each other forming a hairpin-structure, wherein the hairpin structure has a melting temperature (T_PIN), the first signal pin section (5') designed to form a hairpin structure is not complementary to the target nucleic acid oligomers (10), and the signal nucleic acid oligomers (2) are modified in the region of the first signal pin section (5') designed for forming the hairpin structure with at least one redox-active detection label (9); c) preparing a sample having target nucleic acid oligomers (10); d) preparing a reaction solution for performing a nucleic acid amplification, wherein the reaction solution contains at least nucleotides, at least one type of primer (11', 12') and at least one type of nucleic acid polymerase (13) having exonuclease activity; e) mixing the reaction solution prepared in step d) with the signal nucleic acid oligomers (2) prepared in step b) and mixing the sample prepared in step c) with target nucleic acid oligomers (10); f) amplifying the target nucleic acid oligomers (10) by means of nucleic acid amplification to form signal nucleic acid oligomer portions (14); g) bringing the reaction mixture obtained in step f) into contact with the modified surface prepared in step a); h) detecting the signal nucleic acid oligomer portions (14) formed by means of an electrochemical detection method at a temperature T_DET ≤ T_PIN; i) repeating the steps f) to h) multiple times; and k) comparing the different values obtained in step h).

Description

Method for the electrochemical detection of nucleic acid oligomer

hybridization events

Technical area

The present invention relates to a method for the electrochemical detection of nucleic acid oligomer hybridization events.

State of the art

In disease diagnosis, microbiological diagnostics, toxicological testing, genetic research and development, as well as in the agricultural and pharmaceutical sectors, the direct detection of very small amounts of nucleic acids has become indispensable. Increasingly, detection techniques using array technology using so-called DNA chips have become available which enable surface-sensitive detection of nucleic acid oligomer hybridization events. With the help of this sensitive method, nucleic acids can be detected with a very low detection limit. Often, however, prior to the actual detection, a DNA amplification step is necessary in which the DNA molecules to be detected are amplified so that they are ultimately present in a concentration which is above the detection limit of the selected detection method. The PCR is such a basic method for molecular biology, which essentially serves the duplication of DNA molecules and allows the rapid, sensitive direct detection of minute amounts of DNA or RNA. In recent years, this method has conquered the laboratories, as their application is very broad and complex. The PCR has in almost all areas of the Science and medicine, including forensic medicine, prenatal diagnostics, oncology and last but not least in microbiological diagnostics. For example, PCR in the field of clinical diagnostics is usually the method of choice for z. B. detect pathogens. It is also used in the food industry as a detection method for harmful germs.

The PCR is an enzymatic reaction for the amplification of nucleic acid molecules, which essentially takes place in an aqueous or liquid reaction mixture with very small volumes. The reaction mixture contains a nucleic acid-containing sample and the primers, nucleotides and a polymerase which are also necessary for the reaction. By adding buffers and preferably bivalent ions such. B. Mg ²⁺ , the reaction mixture is adjusted so that prevail for the respective application optimum reaction conditions.

The polymerase chain reaction is based on a recurring cycle of three steps occurring at different temperatures, namely denaturation, hybridization and extension.

In the denaturation, the reaction mixture is heated to a temperature greater than 90 ° C, preferably 94 ^< C to 95 ^. As a result, the two complementary strands of a double-stranded DNA separate, the DNA is "melted" or denatured and is then present in single strands.

During the hybridization (also "annealing"), the temperature is lowered to a so-called "annealing temperature". At this temperature, the primers combine with the DNA, they hybridize. At the sites where the primer is bound, the polymerases anneal and begin to elongate by attaching additional complementary nucleotides to the 3'-OH end of the primer to synthesize a counterstrand.

Each repetition of the above three steps doubles the number of copied ones DNA molecules. After 20 cycles, about one million molecules are formed from a single DNA double strand.

The number of cycles can be selected according to the type of application, the nature of the sample and the specific requirements and reaction conditions. Likewise, the setting of the time intervals for the individual steps can be adapted to the requirements of specific types of applications.

Noteworthy in this context is a special type of PCR, the real-time PCR. This quantitative PCR method is becoming increasingly popular in molecular biology laboratories because it allows detection of the sample DNA or amplification products in real time and at the same time the determination of the amount of sample DNA present. The determination of the amount of DNA at the end of a PCR ("end point determination") for many reasons does not allow conclusions about the number of originally present molecules, since, for example, at the beginning and at the end of the PCR conditions for the polymerases are not necessarily optimal Therefore, quantification at the end of a PCR may be very inaccurate, and much more accurate quantification is possible if the number of DNA molecules formed is already detected during the reaction, that is, after each cycle ,

Most known methods for real-time PCR use the fluorescence measurement, sequence-independent and sequence-specific detection methods are known. In the sequence-independent detection methods, for example, an intercalating fluorescent dye is used, which stores sequence-independently in double-stranded DNA. By amplifying a DNA, the total amount of DNA increases, the fluorescence signal increases. For sequence-specific detection methods, very specific targets or gene segments are to be detected and quantified in a single assay. For this purpose, oligonucleotide probes are used which bind to the amplified target DNA. Several principles for the sequence-specific detection of a fluorescence signal and thus for the detection and quantification of the target DNA are available. For example, the detection can be performed via a so-called FRET principle (fluorescence resonance energy transfer principle). In this case, two specific oligonucleotide probes, which can bind directly adjacent in the target used. Each oligonucleotide probe is labeled with a different fluorescent dye, wherein one of the dyes, after excitation with light by its emitted photons again stimulates the second dye if this is in sufficient proximity. The emission light of this second dye is detected by a suitable device. The increase in fluorescence intensity reflects the increase in the target.

Another possibility for the sequence-specific measurement of a fluorescence signal is based on the principle of fluorescence quenching. A specific fluorescence-labeled probe is additionally equipped with a fluorescence quencher that is sufficiently close to the fluorophore that the probe does not fluoresce because of the quenching process. With proper design of the probe, due to hybridization of the probe to the target, the quencher is spatially removed from the fluorophore, quenching is prevented, and fluorescence emission occurs. The quencher can also be cut off in probes bound to the target by means of a suitable exonuclease activity of a polymerase in the PCR approach. Again, the increase in fluorescence intensity reflects the increase in the target.

As an alternative to fluorescence-dependent detection methods, a library of known DNA sequences ("probe oligonucleotides") is fixed in an ordered grid for gene analysis by means of an already mentioned detection method via array technologies on a chip, for example on a surface, so that the position of each individual DNA Sequence is known. If fragments of active genes ("target oligonucleotides") whose sequences are complementary to specific probe oligonucleotides on the chip exist in the assay solution, then the target oligonucleotides can be identified by detection of the corresponding hybridization events on the chip. Methods for surface-sensitive detection of nucleic acid oligomer hybridization events are well known. For example, WO 2003/018834 A2 describes a displacement assay for the detection of nucleic acid oligomer hybridization events. In such an electrochemical process, the association events are evidenced by the change in the electrochemical properties of the probe molecules associated with the association. WO 201 1/069501 A1 combines the high sensitivity of a surface-sensitive detection of nucleic acid oligomer

Hybridization events with a preceding amplification of the target nucleic acid oligomers by a PCR. As a result, the limit for the detection of the target nucleic acid oligomers originally present in the sample can be shifted to very small concentrations.

A disadvantage of this method has been found that the displacement of the signal nucleic acid oligomers hybridized to the probe nucleic acid oligomers by the target nucleic acid oligomers causes a decrease in the signal intensity. The detection of target nucleic acid oligomers thus takes place on the basis of a maximum detection signal by a subsequent decrease in the signal intensity.

It is well-known that the detection of any target molecules can be done with much better detection limit, if the detection of a zero signal emanates, i. in the absence of the desired target molecules no signal is detected. It would be desirable if the signal intensity correlated directly with the number of target molecules present. From the increase in signal intensity, the formation of target molecules could then be followed.

Presentation of the invention It is therefore an object of the present invention to provide a method of detecting nucleic acid oligomer hybridization events which enables the detection of target nucleic acid oligomers by an increase in signal intensity.

This object is achieved by the method according to independent claim 1. Further advantageous details, aspects and embodiments of the present invention will become apparent from the dependent claims, the description, the figures and the examples.

The following abbreviations and terms are used in the context of the present invention:

DNA deoxyribonucleic acid

RNA ribonucleic acid

A adenine

G guanine

C cytosine

T thymine

u uracil

Base A, G, T, C or U

Bp base pair

Nucleic acid At least two covalently linked nucleotides or at least two covalently linked pyrimidine (eg cytosine, thymine or uracil) or purine bases (eg adenine or guanine). The term nucleic acid refers to any "backbone" of the covalently linked pyrimidine or purine bases, such as the sugar-phosphate backbone of the DNA, cDNA or RNA, to a peptide backbone of the PNA or to analogous structures (eg, phosphoramid , Thio-phosphate or dithio-phosphate backbone). An essential feature of a nucleic acid is that it naturally occurring cDNA or RNA can bind sequence-specific.

Nucleotide, nt monomer building block of a nucleic acid oligomer

Oligonucleotide, Ol equivalent to nucleic acid oligomer, e.g. a DNA,

PNA or RNA fragment of unspecified base length.

Sequence Nucleotide Sequence Complementary in a Nucleic Acid Oligomer In order to form the Watson-Crick structure of double-stranded nucleic acid oligomers, the two single strands hybridize, the nucleotide sequence of one strand being complementary to the nucleotide sequence of the other strand, so that the base A (or C) of the one strand with the base T (or G) of the other strand forms hydrogen bonds (in RNA, T is replaced by uracil).

Mismatch To form the Watson-Crick structure of double-stranded nucleic acid oligomers, the two single strands hybridize such that the base A (or C) of one strand forms hydrogen bonds with the base T (or G) of the other strand (in RNA, T is replaced by uracil ). Any other base pairing within the hybrid does not form hydrogen bonds, distorts the structure, and is referred to as a "mismatch."

Perfect Match Hybrid of two complementary nucleic acid oligomers, in which no mismatch occurs.

ss Single Strand (single strand)

ds Double Strand (double strand)

redox-active Designates the property of a unit under certain external circumstances to donate electrons to a suitable oxidant or to take up electrons from a suitable reducing agent.

Linker, Spacer Molecular connection between two molecules or

between a surface atom, a surface molecule or a surface molecule group and another Molecule. As a rule, linkers are commercially available as alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl or heteroalkynyl chain, the chain being derivatized in two places 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 may also be photoactivatable, ie the reactive groups are activated only by light of specific or arbitrary wavelength. Nonspecific nt, ie, nt complementary to other bases, can also be used as linker / spacer, in particular in the case of the attachment of probe oligos to a surface.

The present invention provides a method of detecting nucleic acid oligomer hybridization events, comprising the steps of a) providing a modified surface, wherein the modification consists in the attachment of at least one type of probe nucleic acid oligomers, the probe nucleic acid oligomers forming a first signal pin Have a section of signal nucleic acid oligomers complementary probe pin section,

b) providing at least one type of signal nucleic acid oligomers, wherein

the signal nucleic acid oligomers have a first signal pin section which is complementary to the probe pin section of the probe nucleic acid oligomers,

the signal nucleic acid oligomers have a signal recognition section that is complementary to a target recognition section of target nucleic acid oligomers,

the signal nucleic acid oligomers have a first and a second signal pin section complementary to one another to form a hairpin structure, the hairpin structure having a melting temperature T _P , _N , the first signal pin section is not complementary to the target nucleic acid oligomers, and

the signal nucleic acid oligomers in the region of the first signal pin section are modified with at least one redox-active detection label,

c) providing a sample with target nucleic acid oligomers,

d) providing a reaction solution for carrying out a nucleic acid amplification, wherein the reaction solution contains at least nucleotides, at least one type of primer and at least one type of nucleic acid polymerase with exonuclease activity,

e) mixing the reaction solution provided in step d) with the signal nucleic acid oligomers provided in step b) and the sample provided in step c) with target nucleic acid oligomers,

f) Amplification of the Target Nucleic Acid Oligomers by Nucleic Acid Amplification to Form Signal Nucleic Acid Oligomer

Sections,

g) contacting the reaction mixture obtained in step f) with the modified surface provided in step a),

h) detection of the signal nucleic acid oligomer fragments formed by an electrochemical detection method at a temperature T _DE T ^ T _P | _N , i) multiple repetition of steps f) to h),

k) comparing the different values obtained in step h).

The term hairpin is used in the context of the present invention for a DANN secondary structure in which a hairpin structure is formed by intramolecular base pairings. A hairpin consists of a double-stranded section, which in the present case is also referred to as a "double helix section" or "pin" or "star", and of a single-stranded loop section, which in the present case is also referred to as a "loop".

The hairpin complementary sections of the signal nucleic acid oligomer forming the hairpin double-stranded pin are referred to in the present invention as "first and second signal pin sections." The term "probe pin section" as used herein. describes a portion complementary to the first signal pin portion on the probe nucleic acid oligomer.

In the context of the present invention, the term "sequence sections arranged within the hairpin structure" is understood to mean that the sequence sections in the loop region of the hairpin and thus-in relation to the single strand-are complementary between the two to form a hairpin structure Signal pin sections of the corresponding single strand are arranged.

The term "signal recognition portion" as used in the present invention refers to a portion of the signal nucleic acid oligomer complementary to a portion of the target nucleic acid oligomer and denotes a sequence portion in the signal nucleic acid oligomer which, for example, has a specific sequence portion due to its complementary structure In analogy thereto, the corresponding section of the target nucleic acid oligomer which is recognized by the signal recognition section is referred to herein by the term "target recognition section".

In the context of the present invention, a "substantially complementary structure" is understood as meaning sequence sections in which maximally 10% of the base pairs form mismatches. the base pairs form mismatches.

Of the components provided according to the invention, the signal oligonucleotides are present predominantly in a hairpin structure under normal conditions, the target nucleic acid oligomers form a double strand, the primers are usually single-stranded and the nucleic acid polymerase is usually unbound and has no synthesis activity. The hairpin structure of the Signaloligonukleotide has a melting temperature Τ _Ρ , _Ν . In the context of the present invention, the melting temperature of a hairpin structure is understood to be the temperature at which 50% of the signal oligonucleotides are in the form of hairpin and 50% of the signal oligonucleotides are in open form.

To start an amplification cycle, all potential hybrids / self-hybrids are dissociated by a suitable measure known to the person skilled in the art, for example an increase in temperature. In a subsequent decrease in temperature, the primers hybridize to complementary sites of the still single-stranded targets. In turn, the polymerase binds to the hybrid of primer and target in the region of the 3 ^" - OH end of the primer and begins with the extension of the primer at its 3 ^" OH end. In this polymerization, the present single strand of the target is read as a template and a new double strand is formed.

The hairpin of the Signaloligonukleotids remains during the temperature reduction at least partially in its open form, the signal-recognition section hybridizes to the corresponding target recognition portion of the single-stranded targets and forms with this a double strand.

Since the first signal pin portion of the signal oligonucleotide is not complementary to the target nucleic acid oligomers, it does not hybridize with the target nucleic acid oligomers and therefore exists as the target protruding portion. If the polymerase now arrives at a site of the target on which the signal recognition section of a signal oligos is already bound during the polymerization, the enzyme, because of its exonuclease activity, cuts the first signal pin section not hybridized to the target in a region of the hybridized signal recognition section. This results in a short signal oligonucleotide portion to which the redox-active detection label is bound and which is separated from a remaining signal oligonucleotide trunk, which in turn comprises the second signal pin portion. It should be noted here that the signal oligonucleotide cuts can be cut from both the 3 'end and the 5' end of the signal oligonucleotide because the redox-active detection label can be completely equivalent in the region of the 3 'or the 5' end.

The resulting, with the redox-active detection label modified signal oligonucleotide sections can bind due to the signal pin sections to the complementary probe pin sections of the probe oligonucleotides and thus to the modified surface. The redox-active detection labels are used for the electrochemical detection of signal oligonucleotide segments bound to the modified surface (electrodes) by hybridization with the probe oligonucleotides. When a corresponding voltage is applied to the test site, a current corresponding to the degree of hybridization of probe oligonucleotides and bound signal oligonucleotide sections is measured. This electrically detectable signal is at a temperature T _DE T ^ Τ _Ρ , _Ν significantly higher than the corresponding signal of the original signal oligonucleotides, since the latter are not or only to a very small extent bound to the probe oligonucleotides in the hairpin form.

As the nucleic acid amplification progresses, the replication of the target nucleic acid oligomers proceeds repeatedly to form the signal oligonucleotide portions, and the concentration of free signal oligonucleotide portions increases with increasing amplification, which can be understood at the test site. The increase in concentration leads to an occupied with probe nucleic acid oligomers test site to a - compared to the measurement at cycle 0 - significantly accelerated Anhybridisierung the Signaloligo sections and thus to an increase in the detected electrochemical signal.

The method according to the invention thus provides a method with which the progress of a real-time nucleic acid amplification can be monitored by an increase of the electrochemically detected signal, starting from a substantially zero signal. The electrochemically detected signal increases in proportion to the number of target nucleic acid oligomers. This makes it possible to detect the smallest amounts of target nucleic acid oligomers. Preferably, the signal nucleic acid oligomers are modified with multiple detection labels, thereby obtaining higher intensity signals. According to the invention, the detection label used is a redox-active substance. The signal nucleic acid oligomers preferably have 10 to 200 bases, in particular 20 to 100 bases, particularly preferably 25 to 70 bases. With the help of signal nucleic acid oligomers of this length, all desired targets can be uniquely identified. According to the invention, the amplification of the target nucleic acid oligomers and the detection of the signal nucleic acid oligomer sections are repeated several times, thereby enabling the determination of the increase in signal intensity over time in the course of the amplification of the target nucleic acid oligomers. The concentration of the signal nucleic acid oligomers in the mixture prepared in step e) is preferably between 10 ^{~ 15} mol / l and 10 ^{~ 5} mol / l, more preferably between 10 ^{~ 13} mol / l and 10 ^{~ 7} mol / l and particularly preferred between 10 ^{~ 11} mol / l and 10 ^{~ 7} mol / l. In addition, according to a preferred embodiment of the present invention, the signal nucleic acid oligomers additionally have at least one signal docking section, and the probe nucleic acid oligomers additionally have a probe dock section which is complementary to the signal docking section of the signal nucleic acid oligomers, wherein the signal Dock portion of the signal nucleic acid oligomers adjacent to the first signal pin portion is arranged. More preferably, the probe dock portion of the probe nucleic acid oligomers is located adjacent to the probe pin portion of the probe nucleic acid oligomers. Most preferably, the signal docking portion of the signal nucleic acid oligomers is not complementary to the target nucleic acid oligomers.

The signal docking portion of the signal nucleic acid oligomer is adjacent to the first signal pin portion which is provided with the redox active detection label. In turn, the probe dock portion is adjacent to the probe pin portion of the probe nucleic acid oligomer. In the case of a signal-to-docking section that is not complementary to the target, the latter remains unbound even when the signal recognition section is hybridized to the target together with the first signal pin section, and is substantially "projecting" from the target It is ensured that the signal-nucleic acid oligomer section is thus extended around the signal dock section in addition to the first signal pin section in a signal nucleic acid oligomer section formed by means of the amplification reaction the signal docking portion is complementary to the probe docking portion of the probe nucleic acid oligomers, the potential hybridization region between signal nucleic acid oligomer portion and probe nucleic acid oligomer is extended by these additional signal dock and probe dock portions becomes a faster and more stable binding of subsequently formed signal olig causes onukleotid sections of the probe nucleic acid oligomers and thus increases the detection accuracy.

The fact that the signal dock portion is not complementary to the target also proves advantageous for another reason. Target-complementary signal docking portions could bind to the single-stranded target nucleic acid oligomers. However, since the signal oligonucleotide sections are to be detected electrochemically by binding to the probe nucleic acid oligomers, binding to the target nucleic acid oligomers is undesirable and is advantageously avoided.

Most preferably, the signal docking portion is a variable encoding the signal recognition portion of the signal nucleic acid oligomers. Thus, for example, defined signal dock sections can be coupled in a predetermined manner with a specific signal detection section, so that a specific sequence of the signal dock section corresponds to a specific target sequence.

A parallel detection of different targets using a DNA chip can be carried out in this way very easily. It just has to be one corresponding number of different sequences can be used as signal docking sections of the signal nucleic acid oligomers. Different targets can be detected on a DNA chip which has probe nucleic acid oligomers with a number of correspondingly different probe dock sections at different areas. If different targets are detected in separate experiments, the same sequence of the signal docking portion of the signal nucleic acid oligomers can be used in the different approaches. The signal nucleic acid oligomers are preferably modified in the region of the first signal pin section and / or in the region of the section arranged adjacent to the signal pin section with at least one redox-active detection label. In addition, if the signal nucleic acid oligomers have a signal dock portion located adjacent to the first signal pin portion, the redox active detection label may be equally bound to one of the two portions. After formation of the short signal oligonucleotide sections by the polymerase with exonuclease activity, both sections mentioned are part of the signal oligonucleotide sections which subsequently hybridize to the probe nucleic acid oligomers and thus be detected.

Depending on the nucleotide sequence and the base length of the two sections mentioned, for steric reasons or for reasons of coupling chemistry, one of the two mentioned sections may be better suited for the modification than the other. Depending on the application and depending on the type of signal nucleic acid oligomers, the optimal choice for modification can be made.

To increase the sensitivity of one of the two sections mentioned or even both sections may be modified with several detection label.

Particularly preferably, the two signal-pin sections of the signal nucleic acid oligomers each have 4 to 10 bases, preferably 5 to 8 bases. The stated number of bases ensures, on the one hand, that under normal conditions the predominant part of the signal nucleic acid oligomers is present as a hairpin, and secondly that the melting temperature of the hairpin is in the range of the temperatures used in the nucleic acid amplification. According to another preferred embodiment of the present invention, the signal recognition portion of the signal nucleic acid oligomers is disposed within the loop portion of the hairpin structure between the signal pin portions. Particularly advantageously, the signal recognition section is arranged in the loop area of the hairpin. For the loop area of a hairpin, both the base length and the base sequence offer greater possibilities of variation, as a result of which very different and specific signal recognition sections in different lengths can be integrated into the loop area.

Although binding of the signal nucleic acid oligomers to the target in its open-chain form is desirable, since in this case the polymerase forms signal oligonucleotide segments to a reproducible extent, however, a further advantage of said embodiment is that the loop region is always single-stranded and, therefore, binding of the signal oligomer to the target is possible even when the signal oligomer is hairpin. This applies only if neglecting steric hindrance. Depending on the placement of the recognition sequence within the loop, correct cleavage by the exonuclease activity of the polymerase during amplification is also conceivable in the case of a hybrid of hairpin and target. At the latest in a subsequent denaturation step, the pin portion of the hairpin disintegrates, releasing the signal nucleic acid oligomer portion and being available for detection at the modified surface.

Preferably, the second signal pin portion of the signal nucleic acid oligomers is not complementary to the target nucleic acid oligomers. Since, according to the said embodiment, the second signal pin portion of the signal nucleic acid oligomers is not complementary to the target sequence, the polymerase advantageously can not dock to the signal oligo and from there extend the strand, which could lead to undesirable by-products. Also preferred are embodiments according to which the free end of the second signal pin portion of the signal nucleic acid oligomers is formed by a nucleotide having an inverted nucleoside or a di-desoxy nucleoside. Particularly advantageous according to this embodiment, a chain extension on the signal nucleic acid oligomer is prevented, thereby avoiding the formation of undesirable by-products.

According to a further preferred embodiment of the present invention, the probe nucleic acid oligomers in their probe pin sections and / or in their probe dock sections have at least one nucleotide that is not complementary to the corresponding signal pin sections and signal dock Is sections of the signal nucleic acid oligomers or the signal nucleic acid oligomer sections.

In the method according to the invention, the binding of the signal oligonucleotide sections to the probe oligonucleotides is detected. An interfering background signal can result from the fact that hairpin signal oligonucleotides bind to the probe oligonucleotides. A better discrimination between signal oligonucleotide sections and hairpin signal oligonucleotides can be achieved by having the probe oligonucleotides in the probe pin and probe dock section (s) contain one, two or more bases that are not complementary to the corresponding signal Pin and signal docking portions of the Signaloligonukleotids are. Due to these so-called mismatches, when hairpin signal oligonucleotides and signal oligonucleotide segments are attached to these bases, a so-called "gap" is produced, which has a great negative effect on the hybridization of hairpin signal oligonucleotides, but has little negative effect on the hybridization of signal oligonucleotide segments. Thus, the binding constant of the hairpin signal oligonucleotides to the probe oligonucleotides is significantly more reduced than the binding constant of the signal oligonucleotide portions to the probe oligonucleotides, resulting in an increased difference between the corresponding signal intensities. A corresponding discrimination-enhancing behavior at the probe can also be achieved if the probe in the region (s) complementary to the signal oligonucleotides is one, two or more bases shorter than the corresponding region of the hairpin signal oligonucleotide or signal oligonucleotide segment.

Preferably, the detection of the signal nucleic acid oligomer cuts formed in the method according to the invention by an electrochemical detection method at a temperature T _DET between 20 'C and 60' C, preferably between 30 'C and 50 ° C. At the temperatures mentioned, a particularly accurate and reproducible detection of the signal nucleic acid oligomer sections is possible.

According to a particularly preferred embodiment of the present invention, the amplification of the target nucleic acid oligomers is carried out by a PCR or by an isothermal amplification. In particular, the PCR represents an established method by which a largely error-free amplification of the target nucleic acid oligomers can be ensured. The present invention also encompasses a signal nucleic acid oligomer for use in a method according to the invention, wherein the signal nucleic acid oligomer has a first and a second signal pin section complementary to each other to form a hairpin structure and the signal nucleic acid oligomer in the range of two Forming a hairpin structure to each other complementary signal pin sections is modified with at least one redox-active detection label.

The present invention also encompasses a kit for carrying out a method according to the invention comprising a modified surface as described above, an effective amount of signal nucleic acid oligomers as described above and a reaction solution for carrying out a nucleic acid amplification, wherein the reaction solution comprises at least nucleotides, primers and at least one species of nucleic acid polymerase having exonuclease activity. The Conductive Surface The term "conductive surface" refers to any electrically conductive substrate capable of covalently or by other specific interactions binding derivatized or non-derivatized probe nucleic acid oligomers directly or after appropriate chemical modification.

Any carrier with an electrically conductive surface of any thickness can be used, in particular surfaces of platinum, palladium, gold, cadmium, mercury, nickel, zinc, carbon, silver, copper, iron, lead, aluminum and manganese. Particularly preferred in the context of the present invention, a gold-coated surface is used.

In addition, any doped or non-doped semiconductor surfaces of any thickness can be used. All semiconductors can be used as pure substances or as mixtures. Examples which may be mentioned here are carbon, silicon, germanium, tin, Cu (I) and Ag (I) halides of any desired crystal structure. Also suitable are all binary compounds of any composition and any structure of the elements of groups 14 and 16, the elements of groups 13 and 15, and the elements of groups 15 and 16. In addition, ternary compounds of any composition and any structure of the elements of Groups 1 1, 13 and 16 or the elements of groups 12, 13 and 16 are used. The names of the groups of the Periodic Table of the Elements refer to the 1985 IUPAC Recommendation.

Binding of nucleic acid oligomers to the surface

Methods for immobilizing nucleic acid oligomers on a surface are known to those skilled in the art. The probe nucleic acid oligomers may, for example covalently bound to the surface via hydroxyl, epoxide, amino or carboxy groups of the support material with hydroxy, amino or carboxyl groups naturally present on the nucleic acid oligomer or attached by derivatization to the probe nucleic acid oligomer. Alternatively, thiol-modified probe nucleic acid oligomers can be chemisorptively bound to eg gold surfaces. The probe nucleic acid oligomer can be bound directly or via a linker / spacer to the surface atoms or molecules of a surface. In addition, the probe nucleic acid oligomer can be anchored by the methods customary in immunoassays, for example by using biotinylated probe nucleic acid oligomers for noncovalent 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 the automated solid-phase synthesis or in separate reaction steps. In this case, the nucleic acid oligomer is linked directly or via a linker / spacer with the surface atoms or molecules of a surface of the type described above. This binding can be carried out in various ways known to those skilled in the art. In this context, reference is made to WO 00/42217 A1.

In addition, probe immobilization on a DNA chip is described in P. Liepold, T. Kratzmuller, N. Persike, M. Bandilla, M. Hinz, H. Wieder, H. Hillebrandt, E. Ferrer, G. Hartwich (2008), Analytical and Bioanalytical Chemistry, Vol. 391, pp. 1759-1772, Electrically Detected Displacement Assay (EDDA): A Practica! Approach to Nucleic Acid Testing in Clinical or Medical Diagnosis is described in detail.

Probe, target and signal nucleic acid oligomers

The probe nucleic acid oligomers of the present invention consist of nucleotides in a particular nucleotide sequence (sequence) and are immobilized on a surface. Target nucleic acid oligomers are defined as molecules that are specifically formed with the signal nucleic acid oligomers of a double-stranded hybrid interact. Target nucleic acid oligomers within the meaning of the present invention are therefore nucleic acid oligomers which function as complex binding partners of the complementary signal nucleic acid oligomer. The target nucleic acid oligomers whose presence is to be detected by the present invention have at least one sequence region whose sequence is complementary or at least substantially complementary to a portion of the signal nucleic acid oligomers.

In the context of the present invention, a nucleic acid oligomer or ns-oligomer is a compound of at least two covalently linked nucleotides or of at least two covalently linked pyrimidine (eg cytosine, thymine or uracil) or purine bases (eg adenine or guanine), preferably a DNA , RNA or PNA fragment. The term nucleic acid refers to any "backbone" of the covalently linked pyrimidine or purine bases, e.g. on the sugar-phosphate backbone of the DNA, cDNA or RNA, on a peptide backbone of the PNA or on analogous backbone structures, such as e.g. a thio-phosphate, a dithio-phosphate or a phosphoramide backbone. An essential feature of a nucleic acid according to the present invention is the sequence-specific binding of naturally occurring DNA, or RNA or derived (transcribed or amplified) structures such as cDNA or amplified cDNA or amplified RNA (aRNA).

The signal nucleic acid oligomers in the context of the present invention are nucleic acid hairpin structures. Hairpins are a special form of secondary structure of nucleic acids and result from the fact that a sequence segment of a single-stranded DNA or RNA molecule can fold back onto a complementary region within the same molecule to form a small double-stranded segment It should be noted at this point that hairpins always exist to a certain extent in the closed form, ie the hairpin form and to a certain extent in the open, ie single-stranded form, it turns out a thermodynamic equilibrium.The ratio of closed to open form becomes For example, influenced by the temperature, for example, at the melting temperature T _P | _N each half of the molecules closed or open before. The melting temperature itself, in turn, depends on the sequence of the complementary sections forming the pin or star, their GC content, their length and the like.

Detection Label / Label (Marker Molecule) The signal nucleic acid oligomers are labeled by derivatization with one or more detectable redox-active substances. This label enables the detection of the complexing events between the signal nucleic acid oligomer sections formed in the method according to the invention and the surface-bound probe nucleic acid oligomers.

As a redox label transition metal complexes, in particular those of copper, iron, ruthenium, osmium or titanium with ligands such as pyridine, 4,7-dimethylphenanthroline, 9,10-phenanthrenquinonediimine, porphyrins and substituted porphyrin derivatives can be used. In addition, the use of riboflavin, of quinones such as pyrroloquinolinequinone, ubiquinone, anthraquinone, naphthoquinone or menaquinone or derivatives thereof, of metallocenes and metallocene derivatives such as ferrocenes and ferrocene derivatives, cobaltocenes and cobaltocene derivatives, of porphyrins, methylene blue, daunomycin, dopamine derivatives, hydroquinone Derivatives (para- or ortho-dihydroxy-benzene derivatives, para- or ortho-dihydroxy-anthraquinone derivatives, para- or ortho-dihydroxy-naphthoquinone derivatives) and similar compounds possible. Particular preference is given to using ferrocene or a ferrocene derivative as the redox-active label.

Indirect labels can also be used in the process according to the invention. The term "indirect labels" is understood to mean those in which the actually detectable form of the label is first formed via an enzyme-catalyzed reaction The detectable form of the label can then be detected on the surface Examples of such indirect labels are known to the person skilled in the art from the literature As is known, alkaline phosphatase (AP) may be used as an example called p-aminophenyl phosphate with the substrate. If AP is bound to the signal nucleic acid oligomer as an indirect marker, electrochemical detection of the signal nucleic acid oligomer can be effected by adding p-aminophenyl phosphate at the time of detection. The electrochemically inactive p-aminophenyl phosphate serves as a substrate of the enzyme AP and is converted to p-aminophenol. After diffusion to a conductive surface, p-aminophenol can now be detected electrochemically, since this form of the substrate (ie after conversion at the AP) is electrochemically active. Alternatively, AP can also be used for chromogenic detection (eg with 5-bromo-4-chloro-3-indoxyl phosphate in conjunction with nitroblue tetrazolium chloride).

Surface-sensitive Electrochemical Detection Methods In electrochemical methods, the kinetics of electrochemical processes can be used to distinguish between redox-active detection labels adsorbed to a surface and dissolved in the supernatant. Surface adsorbed detection labels are generally more rapidly electrochemically reacted (e.g., oxidized or reduced) as the redox-active, volume phase detection label since the latter must first diffuse to the (electrode) surface prior to electrochemical conversion. As examples of electrochemical surface-sensitive methods, cyclovalentammetry, amperometry and chronocoulometry are mentioned. The method of chronocoulometry e.g. allows to distinguish near-surface redox-active components of (identical) redox-active components in the bulk phase and is e.g. in Steel, A.B., Herne, T.M. and Tarlov M.J .: Electrochemical Quantitation of DNA Immobilized on Gold, Analytical Chemistry, 1998, Vol. 70, 4670-4677 and references cited therein. The use of chronocoulometry in a displacement assay for the detection of nucleic acid oligomer hybridization events is described in detail in WO 03/018834 A2, which is hereby incorporated by reference.

An electrochemical measurement variant of hybridization events under Use of a DNA chip is described in P. Liepold, T. Kratzmuller, N. Persike, M. Bandilla, M. Hinz, H. Wieder, H. Hillebrandt, E. Ferrer, G. Hartwich (2008), Analytical and Bioanalytical Chemistry , Vol. 391, pp. 1759-1772, Electrically Detected Displacement Assay (EDDA): A Practica! Approach to Nucleic Acid Testing in Clinical or Medical Diagnosis.

According to a preferred embodiment of the present invention, cyclic voltammetry, amperometry, chronocoulometry, impedance measurement or scanning electrochemical microscopy (SECM) are used for electrochemical detection. The methods mentioned allow an accurate and reliable detection of the hybridization events.

All methods described in the context of the present invention can be carried out using DNA chips. In this case, the modified surface has at least 2 spatially substantially separated regions, preferably at least 4 and in particular at least 12 spatially substantially separated regions.

By "spatially substantially separated regions" is meant areas of the surface which are predominantly modified by attachment of a particular type of probe nucleic acid oligomer. Only in areas where two such spatially substantially separated regions are contiguous may a mixture of different types of probe nucleic acid oligomers occur.

Most preferably, the modified surface has at least 32, in particular at least 64, most preferably at least 96 spatially substantially separated areas. The modified surface provided in step a) of the method according to the invention preferably has an area of 1 μm ² to 1 mm ² , more preferably an area of 10 μm ² to 100 μm ² and particularly preferably an area of about 50 μm ² . According to a further, particularly preferred embodiment, in each case one of the spatially substantially separated regions of the surface is bound one respective type of probe nucleic acid oligomer to the surface, the different types of probe nucleic acid oligomers differing in at least one base from one another. This allows the parallel detection of a variety of different types of target nucleic acid oligomers.

A CMOS-based DNA chip for electrochemical detection of hybridization between probe and signal oligonucleotide is e.g. in Augustyniak, M .; Paul, C; Brederlow, Fl .; Persike, N .; Hartwich, G .; Schmitt-Landsiedel, D .; Thewes, R. (2006), Solid State Circuits, 2006 IEEE International Conference Digest of Technical Papers, 59-68 A 24x16 CMOS-Based Chronoculometric DNA Microarray.

Brief description of the drawings

The invention will be explained in more detail with reference to embodiments in conjunction with the drawings. Show it

1 a shows a probe nucleic acid oligomer in a schematic representation;

Figure 1 b is a signal nucleic acid oligomer in a schematic representation.

FIG. 1 c shows a schematic representation of a target nucleic acid oligomer; FIG.

Fig. 1 d primer and polymerase in a schematic representation;

Fig. 2a in a schematic representation of the essential components of

method according to the invention in their hybridization states under normal conditions; 2b is a schematic representation of the essential components of the method according to the invention in its hybridization states at the start of a PCR cycle;

Fig. 2c is a schematic representation of the replication of the target and the formation of signal oligonucleotide sections;

Fig. 3a is a plot of the peak current cyclovoltammetric measurements of a

Ferrocene labels as a function of time at the beginning of the PCR;

Fig. 3b is a plot of the peak current cyclovoltammetric measurements of a

Ferrocene labels as a function of time after the 30th PCR cycle;

4 shows a plot of the peak current of cyclovoltammetric measurements of a

Ferrocene labels 20 seconds after the appropriate PCR cycle, depending on the number of cycles.

Methods for Carrying out the Invention Electrically Detected Real-Time PCR (eRT-PCR)

For a PCR approach Signaloligonukleotide are given, which are on the one hand to probe oligonucleotides immobilized on a test site of a DNA chip, and on the other hand to the target oligonucleotides (a sequence region of the template or PCR to be amplified by PCR section) are complementary.

In FIGS. 1 a to 1 d, a probe nucleic acid oligomer 1, a signal nucleic acid oligomer 2 and the essential remaining components of an electrically detectable real-time PCR are shown schematically. 1 a schematically shows a test site of a microarray with one of the probe oligonucleotides 1 immobilized thereon. The surface 3 of the test site is made of gold. On the Test site is immobilized on Thiolbindungen (-S-) a kind of probe oligonucleotides 1. The probe oligonucleotides 1 comprise the sections spacer 4, probe pin section 5 and probe dock section 6. The probe dock section 6 can also be arranged between probe pin section 5 and spacer 4. In addition, it is possible for probe pin section 5 and probe dock section 6 to overlap, ie have common bases.

The signal oligonucleotides 2 have a so-called hairpin structure at normal conditions (25.sup.-1 bar), which consists of a pin region constructed from a first and a second signal pin section 5 ', 5 "and a loop region In addition to the first signal pin section 5 ', the signal oligonucleotide 2 of the present example additionally has a signal dock section 6' which is complementary to the probe dock section 6 of the probe oligonucleotides 1. The hairpin carries a covalently bound, electrically detectable one Label 9, wherein in the present example the first signal pin section 5 'is marked with the label The first signal pin section 5' is complementary to the probe pin section 5 of the probe oligonucleotide 1. It is also possible to the signal dock section 6 'is not arranged between the signal pin section 5' and the signal recognition section 8, but at the terminal end at the free end of the signal pin section 5 ' Signal pin section 5 'and the signal dock section 6' overlap, so have shared bases.

The loop region of the signal oligonucleotides 2 has a signal recognition section 8 which is complementary to a target recognition section 8 'of the target nucleic acid oligomer 10 (FIG. 1 c). The target nucleic acid oligomer is usually in the form of a double strand and has primer binding sites 1 1, 12, which in turn are complementary to the primers 1 1 'and 12 ^" (Figure 1 d) represented with suitable exonuclease activity.

FIGS. 2a to 2c schematically show the basic features of the course of an electrically detectable real-time PCR in the volume phase. The essential components in their hybridization states under normal conditions (25 ° C, 1 bar) are shown in Fig. 2a. In general, the signal oligonucleotides 2 are present in a hairpin structure, the target 10 as a double strand, the primers 1 1 ', 12' single-stranded and the polymerase 13 in a state not bound to the DNA. A PCR cycle is started by briefly heating the PCR solution to about 95 ° C to dissociate potential hybrid / self-hybrids (see Figure 2b). If the PCR solution is cooled to a suitable temperature (about 70 ° C.), on the one hand the primers 1 1 ', 12' can hybridize with the single-stranded targets 10 and the polymerase 13 can dock, on the other hand the hairpin of the signal oligonucleotide remains at least partially dissociated, and the signal detection section 8 of the signal oligos 2 can hybridize with the target recognition section 8 '. Since the second signal pin section 5 "of the signal oligos 2 is not complementary to the target sequence, this section is always single-stranded, which is why the polymerase 13 can not dock to the signal oligo 2 and extend from there the strand, resulting in undesirable by-products Alternatively or in addition to a non-complementary embodiment of the second signal pin section 5 ", the signal oligo 2 could also have an inverted nucleoside or a di-desoxy nucleoside at the end of the second signal pin section 5" Known measures can be taken to avoid chain extension of the signal oligonucleotide 2.

The temperature suitable for the state sketched in FIG. 2c is essentially of the primer hybridization temperature T _P ,

Signal Oligohybridisierungstemperatur the Signaloligos 2 with the target 10 in the range 8/8 'T _so and the Hairpinschmelztemperatur T _Pin under the buffer conditions of the PCR dependent and is determined in the primer / probe design in silico by appropriate software or empirical values. As a rough rule of thumb, the appropriate temperature T should apply to the process outlined in Fig. 2c: T ~ T _P ~ T _so ~ (>) T _P , _N , where the temperature T should additionally be in a range where the polymerase is high Rates of target replication (typically around 70 'C); T can be optimized accordingly in preliminary tests.

With a suitable construction of the hairpin signal oligonucleotide, T can also be smaller than T _Pin , if it is ensured that at least a fraction of the signal oligos 2 and / or (partially) closed hairpin structure to the at least in the target recognition section 8 'single-stranded target 10 can bind. If the prerequisites outlined in FIG. 2c are given and the polymerase 13 has a suitably suitable exonuclease activity, this replication causes the signal oligonucleotide 2 bound to the target 10 to be replicated in an area in the vicinity of the transition from the signal dock section 6 'to the signal detection section 8 of the Signaloligonukleotids 2 intersect. This results in a short signal oligonucleotide portion 14 which is separated from the signal oligonucleotide trunk 15 (Figure 2c).

The covalently bound redox-active label, in particular ferrocene 9, of the signal oligonucleotides 2 can be used for the electrochemical detection of signal oligonucleotides 2 bound / hybridized to the modified surface 3, namely the test sites (electrodes). Ferrocene is reversibly oxidizable and reducible. When a corresponding voltage is applied to the test site, a current corresponding to the degree of hybridization of probes 1 and bound signal oligos 2 is measured. Accordingly, the Signaloligo sections 14 at a suitable temperature (T _DE T ^ Τ _Ρ , _Ν ) are bound at the test site with probe oligo 1 and generate an electrically detectable signal, which is significantly higher at T _DE T ^ Τ _Ρ , _Ν corresponding signal of the original Signaloligos 2, since the latter is not or only to a very small extent bound to the probe 1 in the hairpin shape. To aid discrimination between hairpin signal oligonucleotide 2 and signal oligonucleotide section 14, probe 1 between first signal pin section 5 and signal dock section 6 may additionally have one, two or more bases which are not complementary to signal oligonucleotide 2 and upon attachment of hairpin signal oligonucleotide 2 and signal oligonucleotide segments 14 form mismatches and / or a so-called "gap", which has a strong negative effect on the hybridization of hairpin signal oligonucleotides 2, but only slightly negative on the hybridization of signal oligonucleotide segment 14. A corresponding discrimination-increasing behavior on the probe 1 can also be achieved if the probe 1 in the region of the first signal pin and / or the signal dock section 5 ', 6' by one, two or more bases shorter than that corresponding portion of hairpin signal oligonucleotide 2 or signal oligonucleotide portion 14.

Table 1 summarizes the primers, probe, signal and target oligonucleotides used for the following eRT-PCR experiments.

Table 1: 5'-3 'sequences of the oligonucleotides outlined in FIGS. 1 and 2.

^* (hybridisable probe sequence in lowercase letters, spacer in capital letters, Gap small and italics, and mismatches small and bold)

As the progress of the PCR reaction progresses the basic features of the amplification shown in FIG. 2 are repeated and the concentration of the free signal oligonucleotide sections 14 increases markedly with increasing number of cycles of the PCR, which can be reproduced at the test site. The increase in concentration leads to a test site occupied by probes 1 (see Figure 1 a) to a - compared to the measurement at cycle 0 (Figure 3a) - significantly accelerated Anhybridisierung the Signaloligonukleotid sections 14 (Figure 3b).

3a and 3b show measurement results of an electrically detected signal change for "real time" tracking of the progress of a PCR reaction in Fig. 3a, the peak current cyclovoltammetrischer measurements of the ferrocene label 9 as a function of time at the beginning of the PCR (cycle 0 ) for two probes (probe_A_1 and probe_A_3, see Table 1) This representation corresponds to the hybridization behavior of the signal oligos 2 and / or signal oligo sections 14, which depends, inter alia, on the concentration of signal oligos 2 or signal oligo sections 14. In FIG 3a is thus the hybridization behavior of the signal oligonucleotide 2 at 40 °, a temperature at which the signal oligonucleotide 2 is present as a hairpin, with which Fig. 3a finally also represents the background signal of the measurement PCR cycle (the Her-2 PCR, see text) compared to the background signal (Probe_A_3 at 0 cycles) In the PCR, the amount of signal oligo segments 14 corresponding to the progress of the amplification was prepared via the exonuclease activity of the Taq polymerase. The hybridization behavior at cycle 30 essentially corresponds to the background signal (from FIG. 3 a) and the signal of the signal oligoparticles 14 formed.

Real-time PCR performed on the example of Her-2 PCR (Her-2 = human epidermal growth factor receptor 2 cDNA)

The sequences of the essential PCR components are listed in Table 1.

The master mix for the PCR reaction for amplifying a target sequence of Her-2 (template amount about 10 ³ copies) contains appropriate primers (10 μΜ), the dNTP mix (per dNTP 25 μΜ) and MgAc (3 mM) in one Standard PCR buffer (5x Bicin buffer, 250mM Bicin / KOH, pH 8.2, 575mM K-acetate, 40% glycerol (v / v), bicine = N, N-bis (2-hydroxyethyl) glycine). To this master mix are the template and signal oligonucleotides (50nM), as well as control signal oligonucleotides (50 nM), which are completely non-complementary to DNA sequences of the template solution given. Immediately before starting the PCR reaction, PCR polymerase with exonuclease activity is added and the solution filled into a PCR cartridge.

Function and structure of the PCR cartridge and a PCR cycler are described in DE 10 2009 044 431 A1. The PCR cartridge is a 20 mm diameter, 1 mm thick cell containing approximately 0.5 mm of hollow tubing for receiving the solution for the PCR reaction. Inside the cartridge is a DNA chip whose sensor field is in fluid contact with the reaction solution via a bridge channel. The chip has a plated-through hole so that the electrical contacting of the DNA chip necessary for the measured value acquisition can be accomplished from outside via spring contacts.

To carry out the PCR, the PCR cartridge is placed in a PCR cycler. The PCR cycler consists of 3 heating blocks made of a good thermally conductive material (eg aluminum). By means of Peltier elements or heating mats, platinum resistance for temperature measurement and suitable control electronics (PID controller), the heating blocks are brought to the temperature necessary for the PCR reaction. Each block contains a gap into which the PCR cartridge is inserted. Above and below the cartridge, another heating block is located in the area of the DNA chip in order to set the chip temperature independently of the temperature for the PCR reaction in the reaction channel. This centric heating block also serves to mediate the rotational movement of the PCR cartridge and the contacting of the DNA chip in the cartridge. By a suitable choice of the block geometry, the area in which the cell has a certain temperature can be set. In the present example, a 2: 1: 1 ratio of 95 ^ range was chosen to be 72 ° C (annealing) and 72 ^< C (elongation) ranges. There is an air gap between the blocks to prevent heat transfer between the blocks. By varying the rotation frequency, the time during which the critical temperatures for the PCR reaction applied to the cartridge can be varied. Before starting the PCR, the reaction mixture is heated in the PCR cycler for 3 min at 95 ° C and then cooled and after slight mixing of the PCR solution by suitable measures to ensure that fresh solution from the PCR channel wets the chip, a first measurement performed on the integrated DNA chip. At one of the provided test sites, the DNA chip carries probes of the Probe_A_3 type which are at least partially complementary to the signal nucleic acid oligomer section 14 and, at another test site, probes which are complementary to the control signal oligonucleotide. The measurement takes place at 40 ° C., ie well below the T _{pin of} the signal oligonucleotide and corresponds to the measured values shown in FIG. 3 a.

After this first normalization measurement, the PCR reaction is started, the heating blocks of the PCR cycler are set to 96 ° C (melting), 72 ° C (annealing) and 72 ^< C (elongation) and the PCR cartridge with a rotational speed of Turned 2 revolutions per minute (ie 2 cycles per minute) in the heating blocks. The nearly centrically aligned sensor field of the DNA chip is held at about 40 ° C via another heating block. During a measurement of the hybridization process on the chip, the PCR cartridge is generally rotated further.

4, the peak currents of the hybridization behavior (compare FIGS. 3a, 3b) are shown in each case 20 seconds after the corresponding PCR cycle as a function of the number of cycles in order to illustrate the amplification profile during the PCR. From about 20 cycles, this peak current increases significantly. This increase is due to the increased formation of Signaloligo sections and thus to increased amplification of the target DNA Her-2. By appropriate standardization and comparison with the control test site, it is also possible not only to prove the presence of the corresponding target sequence, but also to back to the original amount present (quantitative or semiquantitative real-time PCR). LIST OF REFERENCE NUMBERS

1 probe nucleic acid oligomer

2 signal nucleic acid oligomer

3 Surface of the test site

4 spacers

5 probe pin section

5 'first signal pin section

5 "second signal pin section

6 probe dock section

6 'signal dock section

8 signal detection section

8 'target detection section

9 redox-active detection label

10 target nucleic acid oligomer

1 1, 12 primer binding sites

1 1 ', 12' primer

13 nucleic acid polymerase

14 Signal nucleic acid oligomer section 15 Signal nucleic acid oligomer trunk

Claims

claims

A method for the electrochemical detection of nucleic acid oligomer hybridization events comprising the steps

a) providing a modified surface, wherein the modification in the connection of at least one type of probe nucleic acid oligomers (1), wherein the probe nucleic acid oligomers (1) to a first signal pin section (5 ') of signal nucleic acid oligomers (2) have complementary probe pin section (5),

b) providing at least one type of signal nucleic acid oligomer (2), wherein

the signal nucleic acid oligomers (2) have a first signal pin section (5 ') complementary to the probe pin section (5) of the probe nucleic acid oligomers (1),

the signal nucleic acid oligomers (2) have a signal recognition section (8) which is complementary to a target recognition section (8 ') of target nucleic acid oligomers (10),

the signal nucleic acid oligomers (2) have a first and a second signal pin section (5 ', 5 ") complementary to one another to form a hairpin structure, the hairpin structure having a melting temperature T _P | _N ,

the first signal pin section (5 ') designed to form a hairpin structure is not complementary to the target nucleic acid oligomers (10), and

the signal nucleic acid oligomers (2) in the region of the first signal pin section (5 ') designed to form a hairpin structure are modified with at least one redox-active detection label (9),

c) providing a sample with target nucleic acid oligomers (10), d) providing a reaction solution for carrying out a nucleic acid amplification, wherein the reaction solution comprises at least nucleotides, at least one type of primer (11 ', 12') and at least contains a type of nucleic acid polymerase (13) with exonuclease activity,

e) mixing the reaction solution provided in step d) with the signal nucleic acid oligomers (2) provided in step b) and the sample provided in step c) with target nucleic acid oligomers (10),

f) amplification of the target nucleic acid oligomers (10) by means of nucleic acid amplification to form signal nucleic acid oligomer sections (14),

h) detection of the formed signal nucleic acid oligomer sections (14) by an electrochemical detection method at a temperature

i) repeated repetition of steps f) to h),

k) Comparison of the different values obtained in step h).

The method of claim 1, wherein the signal nucleic acid oligomers (2) additionally comprise at least one signal docking portion (6 '), and the probe nucleic acid oligomers (1) additionally comprise a signal docking portion (6') of the signal Nucleic acid oligomers (2) have complementary probe dock portion (6), wherein the signal dock portion (6 ') of the signal nucleic acid oligomers (2) adjacent to the first signal pin portion (5',) is arranged.

The method of claim 2, wherein the signal docking portion (6 ') of the signal nucleic acid oligomers (2) is not complementary to the target nucleic acid oligomers (10).

Method according to one of claims 2 or 3, wherein the signal nucleic acid oligomers (2) in the region of the first signal pin portion (5 ') and / or in the region of the signal adjacent to the signal pin section (5') arranged signal -Dock portion (6 ') are modified with at least one redox-active detection label (9).

5. The method according to any one of claims 1 to 4, wherein the two signal pin sections (5 ', 5 ") of the signal nucleic acid oligomers each having 4 to 10 bases, preferably 5 to 8 bases.

6. The method according to any one of claims 1 to 5, wherein the signal detection section (8) of the signal nucleic acid oligomers (2) within the loop area of the hairpin structure between the signal pin sections (5 ', 5 " ) is arranged.

7. The method according to any one of claims 1 to 6, wherein the second signal pin portion (5 ") of the signal nucleic acid oligomers (2) is not complementary to the target nucleic acid oligomers (10).

8. The method according to any one of claims 1 to 7, wherein the free end of the second signal pin portion (5 ") of the signal nucleic acid oligomers (2) is formed by a nucleotide which is an inverted nucleoside or a di-desoxy nucleoside having.

9. The method according to any one of claims 1 to 8, wherein the probe nucleic acid oligomers (1) in their probe pin sections (5) and / or in their probe dock sections (6) have at least one nucleotide that is not complementary to the corresponding signal pin sections (5 ') and signal dock sections (6') of the signal nucleic acid oligomers (2) or the signal nucleic acid oligomer sections (14).

10. The method according to any one of claims 1 to 9, wherein the detection of the formed signal nucleic acid oligomer sections (14) in step h) by an electrochemical detection method at a temperature T _DET between 20 ° C and 60 ° C, preferably between 30 ° C and 50 ° C takes place.

1 1. Method according to one of claims 1 to 10, wherein the electrochemical detection is performed by amperometry, chronocoulometry, impedance measurement or scanning electrochemical microscopy (SECM).

12. The method according to any one of claims 1 to 1 1, wherein the amplification of the target nucleic acid oligomers (10) in step f) by a PCR or by an isothermal amplification.

13. The method according to any one of claims 1 to 12, wherein the modification of the modified surface provided in step a) consists in the attachment of several types of probe nucleic acid oligomers (1) and the different types of probe nucleic acid oligomers (1) in spatially separated Regions are bound to the modified surface.

14 signal nucleic acid oligomer (1) for use in a method according to claims 1 to 13, wherein the signal nucleic acid oligomer (1) a first and a second to form a hairpin structure mutually complementary signal pin section (5 ', 5 ") and the signal

Nucleic acid oligomer (1) in at least one of the areas of the two signal pin sections (5 ', 5 ") is modified with at least one redox-active detection label (9).

A kit for carrying out a method according to any one of claims 1 to 13 comprising a modified surface as defined in claims 1 to 13, an effective amount of signal nucleic acid oligomers as defined in claim 14 and a reaction solution for performing a nucleic acid amplification as in claim 1 Are defined.