WO2013091614A1 - Procédé de détection électrochimique d'événements d'hybridation d'oligomères d'acide nucléique - Google Patents

Procédé de détection électrochimique d'événements d'hybridation d'oligomères d'acide nucléique Download PDF

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WO2013091614A1
WO2013091614A1 PCT/DE2012/100295 DE2012100295W WO2013091614A1 WO 2013091614 A1 WO2013091614 A1 WO 2013091614A1 DE 2012100295 W DE2012100295 W DE 2012100295W WO 2013091614 A1 WO2013091614 A1 WO 2013091614A1
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nucleic acid
signal
acid oligomers
probe
complementary
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PCT/DE2012/100295
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English (en)
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Gerhard Hartwich
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Friz Biochem Gesellschaft Für Bioanalytik Mbh
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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
    • C12Q1/6825—Nucleic acid detection involving sensors
    • 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
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • the present invention relates to a method for the electrochemical detection of nucleic acid oligomer hybridization events.
  • nucleic acids 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.
  • 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.
  • buffers and preferably bivalent ions such as 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.
  • the reaction mixture is heated to a temperature greater than 90 ° C, preferably 94 ⁇ C to 95 ⁇ .
  • a temperature greater than 90 ° C preferably 94 ⁇ C to 95 ⁇ .
  • the temperature is lowered to a so-called “annealing temperature”.
  • the primers combine with the DNA, they hybridize.
  • 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.
  • sequence-independent and sequence-specific detection methods are known.
  • 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.
  • sequence-specific detection methods very specific targets or gene segments are to be detected and quantified in a single assay.
  • oligonucleotide probes are used which bind to the amplified target DNA.
  • the detection can be performed via a so-called FRET principle (fluorescence resonance energy transfer principle).
  • FRET principle fluorescence resonance energy transfer principle
  • 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.
  • 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.
  • the quencher 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.
  • probe oligonucleotides 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.
  • WO 2003/018834 A2 describes a displacement assay for the detection of nucleic acid oligomer hybridization events.
  • 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.
  • 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.
  • Oligonucleotide Ol equivalent to nucleic acid oligomer, e.g. a DNA,
  • nucleotide Sequence Complementary in a Nucleic Acid Oligomer
  • 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).
  • 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.”
  • 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.
  • 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,
  • 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
  • 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,
  • step 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,
  • step h comparing the different values obtained in step h).
  • 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”.
  • first and second signal pin sections 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.”
  • probe pin section describes a portion complementary to the first signal pin portion on the probe nucleic acid oligomer.
  • 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.
  • signal recognition portion 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
  • target recognition section the corresponding section of the target nucleic acid oligomer which is recognized by the signal recognition section.
  • 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.
  • 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 ⁇ ⁇ , ⁇ .
  • 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.
  • 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.
  • the primers hybridize to complementary sites of the still single-stranded targets.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ ⁇ ⁇ , ⁇ 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.
  • 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 Anhybridmaschine 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.
  • the signal nucleic acid oligomers are modified with multiple detection labels, thereby obtaining higher intensity signals.
  • 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.
  • 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.
  • the signal nucleic acid oligomers additionally have at least one signal docking section
  • 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.
  • the probe dock portion is adjacent to the probe pin portion of the probe nucleic acid oligomer.
  • the 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 onuk
  • 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.
  • binding to the target nucleic acid oligomers is undesirable and is advantageously avoided.
  • the signal docking portion is a variable encoding the signal recognition portion of the signal nucleic acid oligomers.
  • 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.
  • the redox active detection label may be equally bound to one of the two portions.
  • one of the two mentioned sections may be better suited for the modification than the other.
  • the optimal choice for modification can be made.
  • both sections may be modified with several detection label.
  • 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.
  • 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.
  • 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.
  • 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.
  • correct cleavage by the exonuclease activity of the polymerase during amplification is also conceivable in the case of a hybrid of hairpin and target.
  • the pin portion of the hairpin disintegrates, releasing the signal nucleic acid oligomer portion and being available for detection at the modified surface.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • T DET a temperature between 20 'C and 60' C, preferably between 30 'C and 50 ° C.
  • the amplification of the target nucleic acid oligomers is carried out by a PCR or by an isothermal amplification.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • EDDA Electrically Detected Displacement Assay
  • 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.
  • 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.
  • pyrimidine eg cytosine, thymine or uracil
  • purine bases eg adenine or guanine
  • 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
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • AP alkaline phosphatase
  • 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.
  • AP can also be used for chromogenic detection (eg with 5-bromo-4-chloro-3-indoxyl phosphate in conjunction with nitroblue tetrazolium chloride).
  • Electrochemical Detection 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.
  • 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.
  • cyclic voltammetry, amperometry, chronocoulometry, impedance measurement or scanning electrochemical microscopy are used for electrochemical detection.
  • SECM scanning electrochemical microscopy
  • 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.
  • spatially substantially separated regions 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.
  • 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 ⁇ m 2 to 1 mm 2 , more preferably an area of 10 ⁇ m 2 to 100 ⁇ m 2 and particularly preferably an area of about 50 ⁇ m 2 .
  • 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.
  • 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. 1 d primer and polymerase in a schematic representation
  • FIG. 2a in a schematic representation of the essential components of
  • 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
  • oligonukleotide 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.
  • 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 Thiolitatien (-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
  • 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.
  • 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.
  • 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).
  • 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 '.
  • 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
  • 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 .
  • 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.
  • 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.
  • the Signaloligo sections 14 at a suitable temperature (T DE T ⁇ ⁇ ⁇ , ⁇ ) are bound at the test site with probe oligo 1 and generate an electrically detectable signal, which is significantly higher at T DE T ⁇ ⁇ ⁇ , ⁇ 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.
  • 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.
  • FIG. 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.
  • 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)
  • 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.
  • Her-2 human epidermal growth factor receptor 2 cDNA
  • 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.
  • 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.
  • a DNA chip 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.
  • 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.
  • PID controller suitable control electronics
  • the area in which the cell has a certain temperature can be set.
  • a 2: 1: 1 ratio of 95 ⁇ range was chosen to be 72 ° C (annealing) and 72 ⁇ C (elongation) ranges.
  • the rotation frequency 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.
  • 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.
  • 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.
  • the PCR cartridge is generally rotated further.
  • the peak currents of the hybridization behavior 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

Abstract

L'invention concerne un procédé de détection électrochimique d'événements d'hybridation d'oligomères d'acide nucléique, comprenant les étapes consistant à : a) obtenir une surface modifiée, la modification consistant en la liaison d'au moins un type d'oligomères d'acide nucléique sonde (1), les oligomères d'acide nucléique sonde (1) possédant un fragment d'épingle sonde (5) complémentaire d'un premier fragment d'épingle signal (5') d'oligomères d'acide nucléique signal (2), b) obtenir au moins un type d'oligomères d'acide nucléique signal (2), les oligomères d'acide nucléique signal (2) possédant un premier fragment d'épingle signal (5') complémentaire du fragment d'épingle sonde (5) des oligomères d'acide nucléique sonde (1), les oligomères d'acide nucléique signal (2) possédant un fragment d'identification signal (8) complémentaire d'un fragment d'identification cible (8') d'oligomères d'acide nucléique cible (10), les oligomères d'acide nucléique signal (2) possédant un premier et un second fragment d'épingle signal (5', 5") complémentaires l'un de l'autre de manière à former une structure en épingle à cheveux, la structure en épingle à cheveux présentant une température de fusion (TPIN), le premier fragment d'épingle signal (5') destiné à former une structure en épingle à cheveux n'étant pas complémentaire des oligomères d'acide nucléique cible (10), et les oligomères d'acide nucléique signal (2) étant modifiés dans la zone du premier fragment d'épingle signal (5'), destiné à former une structure en épingle à cheveux, avec au moins une étiquette de détection (9) active redox, c) obtenir un échantillon avec des oligomères d'acide nucléique cible (10), d) obtenir une solution réactionnelle pour la réalisation d'une amplification d'acide nucléique, ladite solution réactionnelle contenant au moins des nucléotides, au moins un type d'amorces (11', 12') et au moins un type d'acide nucléique polymérase (13) à activité exonucléase, e) mélanger la solution réactionnelle obtenue à l'étape d) avec les oligomères d'acide nucléique signal (2) obtenus à l'étape b) et l'échantillon obtenu à l'étape c) avec des oligomères d'acide nucléique cible (10), f) amplifier les oligomères d'acide nucléique cible (10) par amplification d'acide nucléique en formant des blocs (14) d'oligomères d'acide nucléique signal, g) mettre en contact le mélange réactionnel obtenu à l'étape f) avec la surface modifiée obtenue à l'étape a), h) détecter les blocs (14) d'oligomères d'acide nucléique signal formés, au moyen d'une méthode de détection électrochimique, à une température TDET ≤ TP|N, i) répéter plusieurs fois les étapes f) à h), k) comparer les différentes valeurs obtenues à l'étape h).
PCT/DE2012/100295 2011-12-19 2012-09-21 Procédé de détection électrochimique d'événements d'hybridation d'oligomères d'acide nucléique WO2013091614A1 (fr)

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