DE102013215168A1 - Method for multiplying nucleic acids - Google Patents

Abstract

A use of one or more nanoparticles (9), which are each conjugated with at least one oligonucleotide (4), for amplifying nucleic acids (1), one or more of the oligonucleotides (4) having at least one primer sequence (5) and another Section extending from the nanoparticle end of the primer sequence in the direction of the nanoparticle, and wherein the further section has at least one abasic modification (7). In addition, a method for amplifying nucleic acids (1) in a sample with a amplification step for amplifying the nucleic acids and a test step for determining the concentration of the products of the amplification reaction, wherein the assay step begins after the end of the amplification step, and wherein at least one part in the assay step the sample are fed into substances or no substances are supplied. In addition, a method for amplifying nucleic acids (1), wherein nanoparticles (8) in a reaction volume (2) by excitation heat to their environment. Finally, a method for amplifying a nucleic acid by means of a polymerase chain reaction in which a cycle consisting of the steps of denaturation, annealing and elongation, is repeated.

Description

Field of the invention

The invention relates to a method for amplifying nucleic acids.

Background of the invention

Methods of amplifying nucleic acids are known in the art. The patent US 4,683,202 discloses a method by which at least one specific nucleic acid sequence can be amplified from a nucleic acid or from a mixture of nucleic acids, each nucleic acid consisting of two separate, complementary strands of equal or unequal length. The method comprises: (a) treating the strands with two primers for each different specific sequence being amplified under such conditions that for each different sequence being amplified, an extension product is synthesized for each primer complementary to that respective nucleic acid strand; said primers are selected to be substantially complementary to different strands of each specific sequence such that the extension product synthesized from one primer, when separated from its complement, serves as a template for the synthesis of the extension product of the other Can serve primers; (b) separating the primer extension products from the templates to which they were synthesized to form single-stranded molecules; (c) treating the single-stranded molecules of step (b) with the primers of step (a) under conditions such that a primer extension product is synthesized using each of the single strands of step (b) as a template. The steps can be performed sequentially or simultaneously. In addition, steps (b) and (c) may be repeated until the desired extent of sequence replication is achieved. In the case that in the method steps (a) and (c) are carried out by means of a polymerase, the method is commonly referred to as polymerase chain reaction (PCR).

In the International Publication WO 2007/143034 A1 discloses methods which should be suitable for carrying out a PCR. The methods may include using an optical radiation source for heating in a PCR. The methods may also include the use of surface plasmon resonance or fluorescence resonance energy transfer to monitor PCR in real time. The methods may further comprise immobilizing a template, primer or polymerase on a surface, such as gold or another surface, that is active with respect to surface plasmon resonance.

The patent application US 2002/0061588 A1 discloses methods for making nucleic acids locally and directly responsive to an external signal. The signal acts only on one or more specific localized portions of the nucleic acid. According to the invention, the signal can alter the properties of a specific nucleic acid and thereby also its function. Accordingly, the invention provides methods that control the structure and function of a nucleic acid in a biological sample without affecting other components of the sample. In one embodiment, a modulator transfers heat to a nucleic acid or part of a nucleic acid, e.g. B. results in that inter- or intramolecular bonds are destabilized and the structure and stability of the nucleic acid changes. Preferred modulators include metal nanoparticles, semiconducting nanoparticles, magnetic nanoparticles, oxide nanoparticles, and chromophores. It is also suggested to use these methods in conjunction with a PCR. In particular, it is proposed to control a PCR reaction with a modulator.

The patent application US 2003/0143604 A1 relates to the use of nanoparticle detection probes for monitoring replication reactions, in particular PCR. Primarily, the patent application is concerned with the use of nanoparticle-oligonucleotide conjugates treated with a protective reagent, such as bovine serum albumin, to quantitatively and qualitatively detect a target polynucleotide. The patent application discloses nucleic acid amplification and detection using gold nanoparticle primers. In a first step, the nucleic acid target is denatured in the presence of the gold nanoparticles to which primers are attached. In a second step, the gold nanoparticles with the primers attached thereto hybridize to the nucleic acid target and a copy of the complementary DNA sequence is generated from the nucleic acid primers attached to the nanoparticles. Steps one and two are repeated and the optical signal produced by the binding of complementary nanoparticle probes that have been amplified is measured.

The post-published patent application DE 10 2012 201 475.6 relates to a method for amplifying nucleic acids. In this process, nanoparticles in a reaction volume excite heat to their environment.

The problem underlying the invention

The object of the invention is to provide an improved use of one or more nanoparticles, which are each conjugated with at least one oligonucleotide, for the amplification of nucleic acids. The invention is further based on the object to provide an improved method for the amplification of nucleic acids.

Inventive solution

In one aspect of the invention, the invention object is achieved by using one or more nanoparticles, each conjugated to at least one oligonucleotide, for amplifying nucleic acids, one or more of the oligonucleotides having at least one primer sequence, and another portion other than the one nanoparticle near the end of the primer sequence in the direction of the nanoparticle, and wherein the further section has at least one abasic modification.

It is an achievable advantage of this embodiment of the invention that in the event that the primer sequence, typically but not necessarily after its elongation, serves as a template for the synthesis of a complementary strand by means of a DNA polymerase, an activity of the polymer is beyond the abasic modification partially or even completely prevented. In other words, it can be avoided that a part of the portion which is beyond the abasic modification, viewed from the primer sequence, is used by the DNA polymerase as a template for the synthesis of a complementary strand.

Thus, an unnecessary length of the synthesis product can be counteracted by this embodiment of the invention. As a result, z. For example, problems can be counteracted by the fact that an unnecessarily long complementary strand might require an increased melting temperature for the dehybridization of the oligonucleotide conjugated to the nanoparticle. Also, z. B. problems are counteracted by the fact that the unnecessarily long complementary strand in the following hybridization steps unnecessarily hybridizes non-specific and thereby suffers the specificity of the amplification process.

The nanoparticles according to the invention are preferably particles which, due to their size, have special optical properties, eg. B. characteristic absorption or scattering spectra, which do not emerge so clearly in the bulk material or not so clearly. The nanoparticles preferably have a diameter between 2 and 500 nm, more preferably between 3 and 300 nm and most preferably between 5 and 200 nm. Preferred nanoparticles have a diameter between 7 and 150 nm. The nanoparticles can be spherical, but in particular also non-globular forms in question, for. B. elongated nanoparticles (nanorods). In a preferred embodiment of the invention, the nanoparticle comprises at least one semiconductor or a metal, preferably a noble metal, for. Gold or silver. In one embodiment, the nanoparticle consists entirely of the metal, in another, the metal forms only a part of the nanoparticle, z. B. his shell. A preferred nanoparticle may be a shell-core nanoparticle. A preferred nanoparticle may have pores on its surface that can be occupied by atoms or molecules having a size and a charge determined by the properties of the pores; more preferably, these atoms or molecules do not attach to the nanoparticle until it is in one Solution is located. According to the invention, the nanoparticle also comprises the atoms and molecules deposited on its surface. Preferred nanoparticles are suitable for absorbing optical energy because of their material absorption or plasmon resonance.

For the purposes of the present invention, the term oligonucleotide encompasses not only (deoxy) -oligo ribonucleides, but also oligonucleotides which contain one or more nucleotide analogues with modifications to their backbone (for example methylphosphonates, phosphorothioates or peptides nucleic acids [PNA]). especially sugar of the backbone (eg 2'-O-alkyl derivatives, 3'- and / or 5'-amino riboses, Locked Nucleic Acids [LNA], Hexitol Nucleic Acids, Morpholinos, Glycol Nucleic Acid (GNA), Threose Nucleic Acid (TNA) or Tricyclo DNA, see the article by D. Renneberg and CJ Leumann, "Watson-Crick base-pairing properties of tricyclo-DNA", J. Am. Chem. Soc., 2002, Vol. 124, pp. 5993-6002 , the related content of which is incorporated herein by reference) or which contain base analogs, e.g. Eg 7- Deazapurines or universal bases such as nitroindole or modified natural bases such as N4-ethyl-cytosine. In one embodiment of the invention, the oligonucleotides are conjugates or chimeras with non-nucleoside analogs, e.g. B. PNA. The oligonucleotides in one embodiment of the invention contain at one or more positions non-nucleosidic units such as spacers, e.g. As hexaethylene glycol or C _n spacer with n between 3 and 6. As far as the oligonucleotides contain modifications, they are chosen so that even with the modification hybridization with natural DNA / RNA analytes is possible. Preferred modifications influence the melting behavior, preferably the melting temperature, in particular in order to distinguish hybrids with different degrees of complementarity of their bases (mismatch discrimination). Preferred modifications include LNA, 8-aza-7-deaza-purines, 5-propynyl-uracil and cytosine and / or abasic breaks or modifications in the oligonucleotide. Further modifications within the meaning of the invention are, for. B. Modifications with biotin, thiol and fluorescence donor and fluorescence acceptor molecules.

An abasic modification within the meaning of the present invention is a portion of the oligonucleotide in which the sequence of nucleotides is disrupted by the introduction of one or more non-nucleotide molecules; in such a way that a polymerase completely or partially breaks off the synthesis of an otherwise completely or partially hybridized, complementary oligonucleotide at the level of this segment, since there is insufficient base complementarity at this segment.

In a further aspect of the invention, the invention object is achieved by a method for amplifying nucleic acids in a sample, the method comprising a amplification step for amplifying the nucleic acids and a test step for determining the concentration of the products of the amplification step, the test steps after the end the replication step begins and wherein the sample or a portion of the sample in the test step substances are supplied. This also includes cases in which the sample or part of the sample of another preparation, eg. B. a test probe containing test preparation supplied.

It is an achievable advantage of this embodiment of the invention that the amplification step and the assay step can take place under different reaction conditions. Thus, advantageously, further reactants which improve the reaction conditions for the test step, for. As a salt, a buffer or a detergent, be added only after completion of the amplification step. Advantageously, it can be avoided that a compromise between the requirements of the duplicating step and the test step must be concluded for the substances in the sample.

A amplification step in the sense of the present invention is a step of the method according to the invention in which nucleic acids present in the sample are amplified. The amplification step may have several sub-steps, in each of which a duplication takes place. For example, as such a sub-step, one pass of a duplication cycle may be considered, as typically repeated in a polymerase chain reaction (PCR). It is possible that the test step followed by the amplification step is followed by a further amplification step. The method may thus comprise one or more amplification steps.

A test step in the sense of the present invention is a step of the method according to the invention, in which the concentration of the products of the amplification step is determined. This too can be done in several substeps. It is possible that the test step is followed by a further amplification step, followed by another test step, so that two test steps are present. The method may thus comprise one or more test steps. The alternating sequence of replication and test steps can be continued so that the method involves many replication steps and many test steps.

In a further aspect of the invention, the invention object is achieved by a method for amplifying nucleic acids in a sample, the method comprising a amplification step for amplifying the nucleic acids and a test step for determining the concentration of the products of the amplification step and wherein the sample in the test step no Substances are supplied. In this case, the test step can follow the duplication step or overlap with the amplification step, even completely overlapping.

With this embodiment of the invention is advantageously achievable that the test step takes place without further steps and without further loss of time already during the amplification reaction or directly after the amplification reaction. Reactants that the reaction conditions for the test step improve, for. As a salt, a buffer or a detergent, are advantageously already included in the reaction volume during the amplification reaction in this embodiment of the invention.

In a further aspect of the invention, the object of the invention is achieved by a method for amplifying nucleic acids, in which nanoparticles transfer heat to their environment in a reaction volume by excitation.

Known methods for amplifying nucleic acids include one or more steps in which at least portions of the sample must be heated. It can be achieved by the invention that the entire reaction volume does not have to be heated in the process for the amplification of nucleic acids. On the other hand, it is possible to heat only specific parts of the reaction volume by excitation of nanoparticles. Thus, it is advantageously possible to heat only those parts of the reaction volume which have to be heated to duplicate the nucleic acids. In this way, heat-sensitive components of the sample can be spared. Local heating may be faster than global heating of the entire reaction volume when less energy needs to be transferred. Thus, it is advantageously possible by the invention to provide a method of amplifying nucleic acids that is faster and requires less energy.

The process according to the invention takes place in a space which is referred to below as the reaction volume. The reaction volume may be surrounded by a reaction vessel. The reaction volume contains a sample in which usually the nucleic acid (s) to be amplified are present. The sample may comprise a liquid, preferably water. The liquid may advantageously serve as a suspension medium and / or solvent for the originals and complements and / or other constituents of the sample.

If heat is transferred to its surroundings by excitation of a nanoparticle, this means according to the invention that energy is transferred to the nanoparticle, whereby the nanoparticle heats its surroundings by the transmission of the energy. In this case, preferably by the excitation of the nanoparticles, the immediate environment of the nanoparticles heated more than the wider environment of the nanoparticles. Usually, the nanoparticles are first heated by excitation and then transfer heat to their environment. However, it is also conceivable that heat is transferred to the environment by the excitation of the nanoparticles without the nanoparticles themselves first being heated. Preferably, the environment of the nanoparticles is a spherical volume 100 times the diameter of the nanoparticle located at its center, more preferably it is 10 times the diameter, most preferably 4 times the diameter, and preferably less than 2 times the diameter.

In a further aspect of the invention, the invention object is achieved by a method for amplifying nucleic acids by means of a polymerase chain reaction (PCR), in which a cycle consisting of the steps denaturation, annealing and elongation is repeated.

If at least two steps of the PCR are performed at different temperatures, it may be necessary to provide one or more heating steps and / or cooling steps in the cycle in which the reaction volume or portions of the reaction volume are heated or cooled. A heating or cooling step may take place before or after any of the denaturing, annealing and elongation steps. In this case, a heating or cooling step typically overlaps with the preceding and / or subsequent denaturing, annealing or elongation step. Preferably, the heating in the or at least one of the heating steps is achieved at least in part by excitation of the nanoparticles and is preferably local heating.

In a PCR, a cycle comprising the steps denaturing, annealing (also called hybridization) and elongation is repeated, preferably in this order. In addition, preferably, each of these steps in all passes is the same length. But this is by no means necessary; so one of the steps or several steps may well have a shorter duration in one run than in another run. The duration t _{c of} a cycle is referred to below as the cycle duration.

In the following, a nucleic acid to be amplified is referred to as an original; another common name is Amplikon. The original is a single strand and can form a duplex in the reaction volume along with its complementary strand, called the complement. In this case, after each pass a resulting copy of the original is an original for the next pass and one resulting copy of the complement is a complement for the next pass. One cycle of the cycle can increase the number of copies of the original and complement. The cycles of the process according to the invention are run through at least in part of the sample.

The denaturation step serves to denature a nucleic acid duplex, i. H. to separate into their two single strands. Thus, in the denaturation step, for. For example, the original may be separated from the complement. Denaturing is also referred to as melting. The denaturation of the nucleic acid duplex is usually thermally induced, i. H. at least a portion of the nucleic acid duplex or whole duplex is exposed to a temperature, called the denaturation temperature, which causes or at least favors separation of the nucleic acid duplexes. The denaturation temperature need not be a fixed temperature, but may also be a temperature interval within which the temperature varies in the denaturation step. On the one hand, the preferred denaturing temperature is chosen so high that nucleic acid double strands can be separated. On the other hand, the preferred denaturation temperature is chosen so low that a DNA polymerase possibly also present in the sample is not significantly damaged. A typical value for the denaturation temperature is 95 ° C.

The reaction volume also preferably contains at least one, more preferably at least two, oligonucleotides termed primers. One of these primers is referred to as a forward primer and another as a reverse primer. The forward primer is complementary to the 3 'end of the original. The reverse primer is complementary to the 3 'end of the complement. Annealing is understood to mean hybridizing the forward primer to the original and the reverse primer to the complement. The annealing step serves to hybridize the forward and reverse primers to their complementary sequences in the original or complement, respectively. Also annealing is usually thermally induced, i. H. at least a portion of the original or complement or the whole original or complement is exposed to a temperature, termed the annealing temperature, which causes or at least favors hybridization of the forward and reverse primers to their complementary sequences in the original or complement, respectively. Like the denaturation temperature, the annealing temperature may be a temperature range within which the temperature varies in the annealing step. The annealing step typically takes place at temperatures of 50 ° C to 65 ° C. The annealing temperature is chosen so that the most specific possible hybridization of the primer can be done.

Hybridization in the context of the present invention means the formation of a double strand from two single strands, each of which may consist of a nucleic acid and / or an oligonucleotide. In suitable reaction conditions, hybridization usually results in the lowest possible energy state that can be achieved by the connection of the two single strands. In other words, under suitable conditions, the two single strands preferably bind to each other in such a way that the greatest possible complementarity (i.e., specificity) is produced with respect to the sequences of the two single strands. If a nucleic acid A is partially complementary to a nucleic acid B, this means that the nucleic acid A in one part is complementary to a part of the nucleic acid B.

The reaction volume also preferably contains a DNA polymerase. The DNA polymerase can synthesize a copy of the complement in an elongation step from the forward primer. Starting from the reverse primer, the DNA polymerase can synthesize a copy of the original. Through the synthesis, the copy of the complement is hybridized with the original and the copy of the original is hybridized with the complement. For the purpose of elongation, the DNA polymerase is exposed to a temperature called the elongation temperature, which allows or at least favors elongation. The elongation temperature can also be a temperature range within which the temperature varies in the elongation step. When using a polymerase from Thermus aquaticus (Taq), an elongation temperature of 72 ° C is typically used. In some embodiments of the PCR, the annealing and elongation temperatures are identical, that is, both steps occur at the same temperature.

Preferred embodiments of the invention

Advantageous embodiments and developments, which can be used individually or in combination with each other, are the subject of the dependent claims.

Preferably, the invention specifically amplifies nucleic acids originating from organisms, including viruses, which may include, for example, genomic DNA, organelle DNA, plasmid DNA, RNA and mRNA.

The invention is particularly well suited for the amplification of nucleic acids shorter than 150 bases, more preferably shorter than 100 bases, more preferably shorter than 80 bases, even more preferably shorter than 60 bases.

In a preferred embodiment of the invention, the nanoparticles are conjugated with oligonucleotides. The nanoparticles form in this way nanoparticle-oligonucleotide conjugates. Thus, it can be achieved with advantage that the oligonucleotides are specifically heated by excitation of the nanoparticles without the entire reaction volume having to be heated. In a particularly preferred embodiment, the nanoparticles are conjugated with primers. Most preferably, the nanoparticles are conjugated with the forward and reverse primers of the PCR. In a preferred embodiment of the invention, one type of nanoparticle-oligonucleotide conjugate has forward primers but no reverse primer and reverse primer on another type but no forward primer.

In another preferred embodiment, a variety of conjugates of nanoparticles and oligonucleotides are conjugated to both forward and reverse primers. In this embodiment, in a PCR, starting from the forward primer on a nanoparticle, a new DNA single strand complementary to the original is written. This new DNA single strand is conjugated to the nanoparticle because the new DNA single strand contains the forward primer. Immediately after writing, the new DNA single strand forms a double strand with the original. In a subsequent denaturation, the new DNA single strand is separated from the original. At an annealing temperature, the new DNA single strand hybridizes with a reverse primer located on the surface of the nanoparticle to form a loop. For hybridization with the reverse primer of the same nanoparticle, only a short distance has to be traveled. For hybridization with a reverse primer on another nanoparticle, a longer pathway must be taken on average at preferred nanoparticle concentrations. Thus, in this embodiment, it can be achieved with advantage that the annealing takes place more quickly and a PCR can be run through more quickly.

In a preferred embodiment, the heat transferred to the environment by the excitation of the nanoparticles is sufficient to dehybridize the oligonucleotides on the surface of the nanoparticles of nucleic acids hybridized to the oligonucleotides. In this embodiment, nanoparticles are conjugated to oligonucleotides and at least a portion of these oligonucleotides are hybridized with at least partially complementary nucleic acids. By excitation of the nanoparticles, thermal energy is transferred to the surrounding water, so that preferably the temperature of the water around the nanoparticles is sufficient to denature the oligonucleotides of the nucleic acids connected to them.

In the present invention, preferably one or more nanoparticles, each conjugated to at least one oligonucleotide, are used to amplify nucleic acids, wherein one or more of the oligonucleotides has at least one primer sequence and another portion extending from the nanoparticle end of the primer sequence Direction of the nanoparticle extends, and wherein the further section has at least one abasic modification.

The preferred abasic modification is located at the primer sequence-facing end of the further portion adjacent to the primer sequence. Advantageously, this embodiment of the invention can partially or even completely inhibit elongation of a complementary strand beyond the primer.

The preferred abasic modification is located 3 'to the primer sequence. With this embodiment of the invention, elongation of the primer sequence in the 3 'direction beyond the abasic modification is advantageously partially or even completely prevented.

The abasic modification is preferably selected from the group comprising: 1 ', 2'-dideoxyribose (dSpacer), triethylene glycol (Spacer9) and hexa-ethylene glycol (Spacer18).

As far as the further section has several abasic modifications, these are preferably arranged directly adjacent to each other. As far as the further section several abasic modification Preferably, each abasic modification is preferably selected from the group 1 ', 2'-dideoxyribose, triethylene glycol and hexa-ethylene glycol.

The further section preferably has at least inter alia the function of a spacer (hereinafter also referred to as spacer sequence and spacer), which establishes a distance between the primer sequence and the nanoparticle or increases such a distance. In other words, the spacer sequence serves as a spacer for the remainder of the oligonucleotide. Because the primer sequence is further spaced apart from the nanoparticles by the spacer sequence, the nucleic acids to be amplified and the DNA polymerases can advantageously find better access to the primer sequences. In a preferred embodiment, after their synthesis, the copies of the original and the complement remain attached to the surface of the nanoparticles via the spacer sequence. In a particularly preferred embodiment, the spacer sequence has a recognition sequence of a restriction endonuclease, so that the synthesized copies of the nanoparticles can be cut off. This happens preferably after the duplication is completed, but can also happen during the duplication. Thus, it is possible with the invention to produce copies of nucleic acids which are freely present in the sample.

In a preferred embodiment of the invention, filling molecules are applied to the nanoparticles. The filling molecules prevent the unwanted aggregation of the nanoparticles in the sample. Thus, the filling molecules serve with advantage the stabilization of the nanoparticles. Through the filling molecules, the charge of the nanoparticles can be modulated. As a result, the salt concentration that is located in the vicinity of the nanoparticles can be adapted so that the DNA polymerase can synthesize as quickly as possible and the process can advantageously be carried out quickly. The filling molecules may consist of oligonucleotides, which are not primers and are preferably shorter than the primers. The filling molecules can also z. B. of polymers such. For example, polyethylene glycol, exist. The filling molecules, in a preferred embodiment, allow the number of primers on the nanoparticles to be reduced and instead use more filling sequences without causing significant sacrifices in the efficiency of the process. In a preferred embodiment of the method, the spacer sequences are at least as long as the filling molecules. As a result, it can advantageously be avoided that the primer sequences are obscured by the filling molecules.

In a preferred embodiment of the invention, the nanoparticles are linked to the oligonucleotides such that covalent bonds with more than one thiol are present between oligonucleotides and nanoparticles. PCR buffers usually contain dithiothreitol, which destabilizes the thiol bond between a gold nanoparticle and an oligonucleotide, and in particular under thermal stress, such as. During denaturing, for example, oligonucleotides may be released from the nanoparticles. Covalent bonds with more than one thiol between the oligonucleotide and nanoparticles can reduce the rate of oligonucleotide release and thus increase the efficiency of the PCR.

In one embodiment of the present invention, replicating nucleic acids in a sample comprises a amplification step to amplify the nucleic acids and a test step to determine the concentration of the products of the amplification step, wherein the assay step begins after the end of the amplification step and wherein the sample in the substance be supplied.

In another embodiment of the present invention, the method of amplifying nucleic acids comprises a amplification step of amplifying the nucleic acids and a step of assaying the concentration of the products of the amplification step, wherein no substances are added to the sample in the assay step. In this case, the test step can follow the duplication step or overlap with the amplification step, even completely overlapping. Preferably, the salt conditions and / or the buffer, most preferably all chemical reaction conditions in the amplification step and in the assay step are the same.

In the invention, in a preferred embodiment, a global temperature of the sample during the test step is different than a global temperature of the replication step. In other words, at least at one point in the test step, the global temperature of the sample is different than the global temperature of the sample at at least one time of the replication step. Particularly preferably, a global temperature of the sample during the test step, particularly preferably at the beginning of the test step, is especially preferably during the majority of the test step, more preferably throughout the test step, different from a global temperature during the amplification step, more preferably at the end of the amplification step, most preferably during the majority of the amplification step, most preferably throughout the amplification step.

It is an achievable advantage of this embodiment of the invention that the amplification step and the assay step can take place under different reaction conditions. Advantageously, it can be avoided that a compromise must be made at global temperature between the requirements of the replication step and the test step.

For the purposes of the present invention, the global temperature is the average temperature of the sample in which the amplification step and the test step are performed. For the purposes of the present invention, the "overwhelming" part of the time of a step means more than 50% of the duration of this step.

In one embodiment of the invention, "different to" is specifically "higher than", in another embodiment "lower than". In a preferred embodiment, "different" means that the temperatures are more than 1 degree, more preferably more than 2 degrees, most preferably more than 5 degrees, more preferably more than 10 degrees, most preferably more than 20 degrees, most preferably more differ as 40 degrees.

In the invention, in a preferred embodiment, a global temperature of the sample during the test step is substantially equal to a global temperature of the replication step. In other words, at least at one point in the test step, the global temperature of the sample is substantially equal to the global temperature of the sample at at least one time of the replication step. More preferably, a global temperature of the sample during the test step, more preferably at the beginning of the test step, most preferably during the majority of the test step, most preferably throughout the test step is substantially equal to a global temperature during the amplification step, most preferably at the end of the test step Reproduction step, more preferably during the majority of the amplification step, most preferably during the entire amplification step.

In a preferred embodiment, "equal" means that the temperatures are less than 20 degrees, more preferably less than 10 degrees, more preferably less than 5 degrees, more preferably less than 5 degrees, most preferably less than 2 degrees, most preferably less differ as 1 degree.

Preferably, in the test step for determining the concentration of the products of the amplification step, test probes comprising a nanoparticle are used. The test probes are supplied to the sample in one embodiment of the invention in the test step. In another embodiment of the invention, the test probes are supplied to the sample prior to the testing step, preferably before or during the amplification step.

In a preferred embodiment of the method, the oligonucleotides have a spacer sequence as partial sequence on the nanoparticles of the test probes. The spacer sequence lies on the side of the respective oligonucleotide facing the nanoparticle. Thus, the spacer sequence serves as a spacer for the remainder of the oligonucleotide. In a preferred embodiment, an oligonucleotide contains both a partial sequence referred to as a test sequence and a partial sequence which is a spacer sequence. In a preferred embodiment, filling molecules are applied to the nanoparticles. The test sequences can hybridize with products of the amplification reaction. The test sequences are preferably complementary to the products of the amplification reaction.

In a preferred embodiment, the test sequences at the 3 'end carry one or more terminating modifications such as. B. dideoxycytidine (ddC). These modifications can advantageously prevent the 3 'extension of the test sequence by the polymerase and thus prevent the test sequences can serve as primers.

In a preferred embodiment of the invention, nanoparticles which are used for amplification (also referred to as first nanoparticles in order to distinguish them from the nanoparticles of the test probes, also referred to as second nanoparticles hereinafter) are conjugated with forward primers. In the presence of the original and a DNA polymerase, the forward primers are extended to produce complements bound to the first nanoparticles via the forward primers. A complement consists of the forward primer and an extension sequence, which can be extended by extending the Forward primer arises. In an optional intermediate step, the originals are denatured by the complements by local or global heating. The first nanoparticles are then combined with test probes, if not previously done. The test sequences of the test probes are complementary to the extension sequences, so that the test probes can bind via test sequences to the extended forward primer on the first nanoparticles. Under suitable reaction conditions, the compound of the first nanoparticles with the test probes is formed to the extent that nanoparticle-bound complements are present. This means that if no extension sequences arise, no connection of test probes and first nanoparticles arises. Particularly preferably, the reaction conditions of the amplification and the detection according to the invention are selected by test probes such that the extent of the connection of first nanoparticles with test probes allows conclusions to be drawn about the concentration of the original prior to amplification in the sample. By connecting the first nanoparticles with the test probes, a measurable change can occur, eg. B. a redshift or broadening of the plasmon resonance in the extinction spectrum. In a most preferred embodiment, the measurable change produced by the combination of test probes and first nanoparticles is proportional to the concentration of the original in the sample prior to duplication. So can be done with simple means a proof of concentration advantage.

In a further preferred embodiment, the method comprises forward primers which are conjugated with first nanoparticles and free, that is not nanoparticle-bound, reverse primers. In a first step, the forward primers are extended in the presence of the original by a DNA polymerase to nanoparticle-bound complements. In a second step, starting from the free reverse primer that binds to the nanoparticle-bound complement, a copy of the original is synthesized. Thereafter, the first nanoparticles are combined with test probes, if not previously done. The test sequences in this embodiment are complementary to the forward primers. If the forward primers were not extended then the test probes can bind well to the first nanoparticles. If the forward primers have been extended then binding of test sequences to forward primers is hindered by steric hindrance. When a newly synthesized copy of the original is hybridized with the extended forward primer, binding of the test sequence to the extended forward primer is prevented. In this way, the extent of linkage between first nanoparticles and test probes decreases to the extent that products of the amplification reaction, that is, complements and copies of the original, have been synthesized. With a suitable choice of the reaction conditions, a proof of concentration of the original in the sample can be carried out, so that the more original that was present in the sample before the duplication, the lower the measurable change. The measurable change can be z. B. be a redshift or broadening of the plasmon resonance in the extinction spectrum. Thus, a simple test can be designed with advantage, which allows the determination of concentrations of specific nucleic acids.

In a further preferred embodiment, the reverse primers are also conjugated with nanoparticles. In a particularly preferred embodiment, the reverse primers are also conjugated to the first nanoparticles, which are also conjugated to the forward primers.

Preferably, the nanoparticles of the test probes have a different size than the nanoparticles used in the amplification step for amplifying the nucleic acids. Particularly preferably, the nanoparticles of the test probes are smaller than the nanoparticles which are used in the amplification step for amplifying the nucleic acids. Particularly preferably, the volume of the nanoparticles of the test probes is less than 50%, particularly preferably less than 25%, particularly preferably less than 12.5%, particularly preferably less than 6%, particularly preferably less than 3%, particularly preferably less than 1%, most preferably less than 0.1% of the volume of nanoparticles used in the amplification step to amplify the nucleic acids.

By this embodiment of the invention, a different local temperature can be achieved around the nanoparticles of the test probes during the amplification reaction than the nanoparticles used to amplify the nucleic acids. For example, with test probes smaller than the nanoparticles used to amplify the nucleic acids, it can be achieved that, although the first nanoparticles reach a sufficient temperature for the amplification reaction, the test probes reach a lower temperature. This achieves that the test probes are less thermally stressed. It is also achievable that no replication reaction can take place on the test probes. The latter is particularly advantageous in embodiments of the invention in which the test probes are in the sample during the amplification reaction.

In one embodiment of the invention, nanoparticles in a reaction volume by excitation transfer heat to their environment.

The excitation of the nanoparticles preferably locally heats the surroundings of the nanoparticles, since particularly rapid changes in temperature are possible above all when the heated volume accounts for only a small fraction of the total volume. On the one hand, a high temperature difference can already be generated by irradiation with a small energy input. On the other hand, a very rapid cooling of the heated volume is possible if a sufficiently large cold temperature reservoir is present in the irradiated volume in order to cool the nanoparticles and their surroundings again after the irradiation. This can be achieved by irradiating the nanoparticles with sufficient intensity (to achieve the desired temperature rise) and sufficiently short (so that the heat remains localized). In the case that the duplication by means of a polymerase, z. As a DNA polymerase, it is by the local heating, it is possible to expose the polymerases of lower heat.

A local heating in the sense of the present invention is present if the duration of the excitation in the respective irradiated volume (eg in the laser focus) t is shorter or comparably short of a critical excitation duration t1. Here, t1 is given by the time it takes for the heat to diffuse from one nanoparticle to the next at an average nanoparticle distance multiplied by a scaling factor s1, namely at a mean nanoparticle distance | x | and a thermal diffusivity D of the medium between the nanoparticles t1 are given by t1 = (s1 · | x |) ² / D, wherein the thermal diffusivity D typically in aqueous solution has a value of D = 10 ^-7 m ² / s.

The scaling factor s1 is a measure of how far the heat front of a particle propagates during the excitation period. The increase in temperature by an excited nanoparticle at a distance of a few nanoparticle diameters is only a very small fraction of the maximum temperature increase on the particle surface. In one embodiment of the invention, an overlap of the heat fronts of some nanoparticles is permitted in the sense that a scaling factor s1 greater than 1 is used to define the critical excitation duration t1 on the basis of the formula given above. In another embodiment of the invention, no overlap of the heat fronts during the excitation period is allowed (corresponds to highly localized heating) in the sense that a scale factor s1 less than or equal to 1 is used to define the critical excitation duration t1 using the formula given above. For the definition of the local heating, preferably s1 = 100, preferably s1 = 30, preferably s1 = 10, preferably s1 = 7, preferably s1 = 3 and very particularly preferably s1 = 1, preferably s1 = 0.7, preferably s1 = 0, third

Values for s1> 1 can u. a. for example, be advantageous in those cases in which the irradiated volume has a high aspect ratio (as in the focus of a moderately focused laser beam), so that comparatively many nanoparticles are located on the surface of the irradiated volume and therefore less heated nanoparticles are in their environment, and a strong heat outflow from the irradiated volume takes place, so that the heating contribution of the more distant neighbors remains longer negligible.

This means that at a nanoparticle concentration of 1 nM, for example, a mean nanoparticle distance of | x | = 1.2 microns, so that a local heating according to the invention is present, provided that the excitation duration is less than t1 = 14 microseconds (the scaling factor is chosen here s1 = 1).

It can be assumed that, when t> t1 is chosen, the heat given off by the nanoparticles can thus cover a distance greater than the average nanoparticle distance by diffusion during the irradiation, and as a result to a superposition of the nanoparticles Thermal fronts of many nanoparticles leads, so that in the entire volume between the nanoparticles, a temperature increase takes place; the temperature increase in the irradiated volume should be spatially homogeneous the more it is heated, since not only the contributions of the next nanoparticles, but also of more distant neighbors in the temperature distribution around a nanoparticle around. Therefore, when the reaction volume is irradiated with radiation absorbed by the nanoparticles for longer than t1, the heating is said to be global. A global heating according to the invention can, for. B. also take place in that the reaction volume is heated from the outside with a Peltier element or a resistance heater. The global heating can also be done by z. B. the reaction volume is irradiated with a radiation that is absorbed by the water in the sample stronger than or similar to strong as from the nanoparticles.

Temperature increase here means the difference between the temperature at a location at the observation time immediately after the excitation and the temperature at the same location at the time immediately before the excitation. Global heating and local heating can also be performed simultaneously.

The excitation of the nanoparticles is preferably carried out by an alternating field, particularly preferably by an electromagnetic alternating field, most preferably optically. Preferably, the excitation occurs in the far infrared to far ultraviolet light range (in a range of 100 nm to 30 μm wavelength), more preferably in the range of near infrared to near ultraviolet (in a range of 200 nm to 3 μm wavelength ), most preferably by visible light (in a range of 400 nm to 800 nm wavelength). This can offer the advantage over the conventional global heating of the reaction vessel from the outside that does not have to overcome the thermally insulating wall of the reaction vessel, since the energy is transferred directly to the nanoparticles. Thus, a faster heating of the desired portion of the sample can be achieved.

In a preferred embodiment of the invention, the nanoparticles are excited by a laser. More preferably, the laser light has a frequency that excites the surface plasmon resonance of the nanoparticles. The laser can pulsate the light or provide it continuously. If the laser is pulsed, then nanosecond lasers are preferably used. The laser can z. Example, a gas laser, a diode laser or a diode-pumped solid-state laser.

The duty cycle is the ratio of the time interval of the excitation to the duration of a PCR cycle. The duty cycle is preferably chosen so large that the excitation leads to a sufficient denaturation of the DNA double strands by local heating. At the same time, the duty cycle is preferably selected so that the average temperature increase of the entire sample is kept sufficiently small, so that no disturbing influences on hybridization, elongation and denaturation occur. Preferably, the duty cycle for the irradiated volume is less than 50%, more preferably less than 20% and most preferably less than 1%. The duty cycle in the irradiated volume is suitably more than 10 ^-12 , preferably more than 10 ^-10 , more preferably more than 10 ^-9 and most preferably more than 10 ^-8 .

For the purposes of the present invention, the power density is the optical power per area of the light incident on the sample; if it is a pulsed light source, the peak power is decisive. The power density at which the nanoparticles are excited is preferably at least one pass of the cycle, more preferably at least 10 passes of the cycle, more preferably at least 20 passes of the cycle, most preferably at least 40 passes of the cycle, most preferably at least 80 cycles, and more preferably at least 160 passes of the cycle more than 10 W / mm ^2, more preferably more than 50 W / mm ² , more preferably more than 100 W / mm ² , particularly preferably more than 200 W / mm ² , more preferably more than 300 W / mm ² and most preferably more than 400 W / mm ² . With this embodiment of the invention is advantageously achievable that the nanoparticles are heated sufficiently by the excitation.

The power density at which the nanoparticles are excited is preferably less than 20,000 kW / mm ² , more preferably at least one pass of the cycle, more preferably at least 10 passes of the cycle, most preferably at least 20 passes of the cycle, most preferably at at least 40 passes of the cycle, more preferably at least 80 passes of the cycle, and most preferably at least 160 passes of the cycle less than 10,000 kW / mm ^{2, more} preferably less than 5,000 kW / mm ² , most preferably less than 3,000 kW / mm ² , more preferably less than 1000 W / mm ² , more preferably less than 500 kW / mm ² , more preferably less than 300 kW / mm ² , even more preferably less than 150 kW / mm ² and even more preferably less than 80 kW / mm ² . With this embodiment of the invention, damage to the nanoparticles or the DNA bound thereto can advantageously be counteracted or prevented. In a further preferred embodiment, the energy of the light is transferred to it by the material absorption of the nanoparticles. The light used to excite the nanoparticles may also be e.g. B. from a thermal radiator, z. As a flash lamp, come. In a further preferred embodiment of the invention, the nanoparticles are excited by an electromagnetic alternating field or electromagnetic waves which generate eddy currents in the nanoparticles. It is also possible with suitable design of the nanoparticles to excite the nanoparticles with ultrasound

For the purposes of the present invention, the exposure time t _{A is} the total duration in which a source of energy for the purpose of denaturation, for. For example, during the cycle of the PCR, a power suitable for denaturing is applied to a point in the sample to cause heating in the sample.

The energy source transmits a power suitable for denaturation to the said point during the entire time t _A. For example, a laser-powered energy source could be used at higher power for denaturation and subsequent absorbance measurement at lower power. In this case, t _{A is} merely the time the laser transmits the higher power suitable for denaturation to the point.

If multiple power sources are used for denaturing, so refers to the time t _A is preferably in which all energy sources for denaturing at the same time acting on the point. When activating several energy sources, it is often only possible to effect the simultaneous effect of denaturation.

The said point is determined within the part of the sample in which the method is carried out so that t _A assumes the largest possible value. Thus, for example, if the heating is caused by a fixed Peltier element, t _{A is} the total time that heat from the Peltier element flows to that point during that cycle (typically about the duty cycle during the heating step, in each case shorter than the cycle time ). When the heating is caused by a light beam of diameter d, which is scanned at a velocity v through the sample volume, t _{A is} the time duration d / v during which the light beam acts on a point in the sample. When the heating is caused by a pulsed light source whose light beam is not moved relative to the sample during the pulse duration, the pulse duration is the exposure time. When the heating is caused by a pulsed light source being scanned by the sample, the shorter of the two durations (pulse duration and duration d / v ) the exposure time.

The exposure time t _A is at least one pass of the cycle, more preferably at least 10 passes of the cycle, more preferably at least 20 passes of the cycle, more preferably at least 40 passes of the cycle, most preferably at least 80 passes of the cycle, and more particularly preferably at least 160 passes of the cycle, preferably more than 1 ps (picosecond), more preferably more than 30 ps, more preferably more than 100 ps, more preferably more than 300 ps, even more preferably more than 1 ns (nanosecond), most preferably more as 10 ns, particularly preferably more than 100 ns, particularly preferably more than 300 ns, particularly preferably more than 1 μs (microseconds), particularly preferably more than 3 μs, particularly preferably more than 10 μs.

The exposure time t _A is at least one pass of the cycle, more preferably at least 10 passes of the cycle, more preferably at least 20 passes of the cycle, more preferably at least 40 passes of the cycle, most preferably at least 80 passes of the cycle, and more particularly preferably at least 160 passes of the cycle, preferably less than 10 s (seconds), more preferably 1 s, more preferably less than 100 ms (milliseconds), particularly preferably less than 10 ms, particularly preferably less than 1 ms, more preferably less than 500 μs , more preferably less than 100 microseconds, more preferably less than 50 microseconds, most preferably less than 10 microseconds. Preferably, the exposure time t _{A is} shorter than it takes on average until the heat that arises in the vicinity of the nanoparticles, diffuses the average particle spacing, so that on average no significant overlap of the heat fronts of adjacent particles takes place.

More preferably, the exposure time t _{A is} at least one cycle of the cycle, more preferably at least 10 passes of the cycle, more preferably at least 20 passes of the cycle, more preferably at least 40 passes of the cycle, more preferably at least 80 passes of the cycle and most preferably at least 160 passes of the cycle chosen so that the temperature increase around each irradiated nanoparticle on average at a distance of 20 nanoparticle diameters, more preferably 2 nanoparticle diameters, most preferably 1 Nanopartikeldurchmesser, less than half the temperature increase on the surface the nanoparticles fall off.

In one embodiment, a short irradiation time per volume is preferred so that a dehybridized single strand of DNA diffuses away from the nanoparticle during denaturation only less than 100 nm (nanometers), more preferably less than 20 nm, most preferably less than 5 nm can. This is a high probability that the dehybridized DNA single strand binds to an oligonucleotide on the same nanoparticle ("rehybridization"). This may allow for an accelerated process according to the invention. For this purpose, exposure times t _A between 0.1 ns and 1000 ns are preferable, in particular between 1 ns and 300 ns.

In a preferred embodiment for rehybridization, the concentration of primer-conjugated nanoparticles is less than 10 nM, wherein the exposure time t _{A is} preferably between 1 ns and 10 microseconds (microsecond), more preferably between 10 ns and 1 microseconds, and most preferably between 15 ns and 300 ns. The time interval of the excitation is preferably not selected to be significantly smaller than 1 ns, since otherwise the time of heating the DNA double strand is insufficient for the two single strands contained to be sufficiently separated from one another by diffusion, so that they do not immediately hybridize again.

The irradiation times per sample volume (ie the time that a certain volume per cycle is irradiated optothermally for heating) are preferably below 1 s / μl, particularly preferably below 0.1 s / μl, or 0.01 s / μl, respectively less than 0.001 s / μl. Preferably, the irradiation times per volume, depending on the radiation source used, are simultaneously greater than 1 ps / μl, or preferably greater than 10 ps / μl, or 100 ps / μl (eg when pulsed lasers are used) other embodiments (eg lasers in CW mode) greater than 10 ns / μl, or 100 ns / μl.

The heating time is at least one pass of the cycle, more preferably at least 10 passes of the cycle, more preferably at least 20 passes of the cycle, more preferably at least 40 passes of the cycle, most preferably at least 80 passes of the cycle, and most preferably at at least 160 passes of the cycle preferably less than 100 ms, more preferably less than 10 ms, particularly preferably less than 1 ms, particularly preferably less than 100 μs, particularly preferably less than 50 μs, particularly preferably less than 10 μs, particularly preferably less than 5 μs, more preferably less than 1.5 μs.

The heating time is at least one pass of the cycle, more preferably at least 10 passes of the cycle, more preferably at least 20 passes of the cycle, more preferably at least 40 passes of the cycle, most preferably at least 80 passes of the cycle, and most preferably at at least 160 passes of the cycle, preferably more than 1 ns, particularly preferably more than 5 ns, particularly preferably more than 10 ns.

The cooling time is at least one pass of the cycle, more preferably at least 10 passes of the cycle, more preferably at least 20 passes of the cycle, more preferably at least 40 passes of the cycle, most preferably at least 80 passes of the cycle, and most preferably at at least 160 passes of the cycle preferably less than 100 ms, more preferably less than 10 ms, particularly preferably less than 1 ms, particularly preferably less than 100 μs, particularly preferably less than 50 μs, particularly preferably less than 10 μs, particularly preferably less than 5 μs, more preferably less than or 1.5 μs.

The cooling time is at least one pass of the cycle, more preferably at least 10 passes of the cycle, more preferably at least 20 passes of the cycle, more preferably at least 40 passes of the cycle, most preferably at least 80 passes of the cycle, and most preferably at at least 160 passes of the cycle, preferably more than 1 ns, particularly preferably more than 5 ns, particularly preferably more than 10 ns.

The heating-up time is the time that elapses after the excitation intensity I (t) of the light source has reached its maximum value in the respectively excited volume until at each point in the excited volume a temperature is established that does not exceed a maximum of 3 ° even if the exposure time is doubled C changes.

The cooling time is the period of time after the switch-off time of the exciting light source, which elapses until at each point in the considered volume a temperature is set that deviates by a maximum of 3 ° C from the temperature before the action.

The switch-off time t _{off of} the exciting light source is defined as the time at which the excitation intensity I in the considered volume has decreased to less than 5% of the maximum excitation intensity (eg after the pulse of a laser).

Determination of the heating and cooling time: The temporal temperature development at distance r from the center of a nanoparticle with radius rNP is obtained by numerically solving the heat equation in a sufficiently large water sphere with radius rMax around the nanoparticle, whereby the nanoparticle itself is taken out of the simulation range. By exploiting the ball symmetry, a one-dimensional radial heat equation results in the range rNP to rMax, t> 0: α / r²∂ _r (r ² · ∂ _r T (r, t)) = ∂ _t T (r, t), where T (r, t) is the temperature at point r at time t and α denotes the thermal diffusivity of the water (α = 1.43 · 10 ^-7 m ² / s).

As a starting condition, the temperature of the surrounding medium is set to T ₀ before the optical excitation: T (r, 0) = T ₀ .

The boundary conditions at the positions rNP and rMax are set as follows: At the position r = rNP, the slope of the temperature profile at time t is obtained from the absorbed power of the nanoparticle at time t (Neumann boundary condition): ∂ _r T (rNP, t) = P (t) / (4 · π · rNP ² · k), where P (t) is the power absorbed by the nanoparticle and k is the thermal conductivity of water (k = 0.6 W / (m · K)). The absorbed power is calculated from P (t) = I (t) · σ, with I (t) the time-dependent excitation intensity of the light source and the absorption cross-section of the particle σ. For example, (i.e., I (t) would be a constant for a CW laser as long as the focus size is not changed, and for a pulsed laser I (t) would represent the time-dependent pulse shape).

At temperature rMax the temperature is kept constant (T (rMax, t)) = T ₀ , Dirichlet boundary condition). For rNP <100 nm, for example, one chooses rMax ≥ 10000 nm. The thermal diffusivity and thermal conductivity of the water is assumed to be constant. In general, α = k / (C · ρ), where C is the specific heat capacity and ρ is the density of water.

By means of suitable programs for the numerical solving of such partial differential equations (eg with the command NSolve in Mathematica, etc.) the above-described heat conduction equation can then be solved and values for the temperature as a function of location and time, T (r, t ).

For example, for a spherical gold nanoparticle with rNP = 30 nm, which is excited at a constant intensity of 1 kW / mm ² at 532 nm wavelength for a period of 100 ns, the following values are obtained for a starting temperature of T ₀ = 30 ° C. T (r = 30 nm, t = 20 ns) = 70 ° C, T (r = 30 nm, t = 100 ns) = 78 ° C, T (r = 30 nm, t = 120 ns) = 36 ° C, T (r = 40 nm, t = 20 ns) = 56 ° C, T (r = 40 nm, t = 100 ns) = 64 ° C, T (r = 40 nm, t = 120 ns) = 36 ° C.

To determine the heating time according to the invention T (r, t) is evaluated for different times. The heating time is then the shortest time _t for true | T (r, t _on) - T (r, 2 * t _on) | ≤ 3 ° C with r ε [rNP; rMAX], ie the amount of the difference of the temperature distribution for the times t _on and 2t _on must be less than 3 ° C for all points outside the nanoparticle.

The cooling time is given as the difference t _x - t _off , where t _{x is} the shortest time for which | T (r, t _x ) -T ₀ | ≤ 3 ° C with r ε [rNP; rMAX] and t _x > t _off .

Preferably, the concentration of the amplicon to be amplified in the process at the beginning of the process is greater than 10 ^-23 M (mol / liter), more preferably greater than 10 ^-21 M, particularly preferably greater than 10 ^-20 M, especially preferably greater than 10 ^-19 M. By this embodiment of the invention can be advantageously achieved that the duplication is sufficiently sensitive to produce a suitable amount of detection amplification products.

Preferably, the concentration of the amplicon to be amplified in the process at the beginning of the process is less than 1 nM (nanomoles / liter), more preferably less than 30 pM (pico moles / liter), even more preferably less than 900 (femtomoles / liter ), more preferably less than 800 fM (femtomole / liter), more preferably less than 500 fM (femtomole / liter), more preferably less than 100 fM, more preferably less than 30 fM. By this embodiment of the invention can be advantageously prevented that the duplication reached saturation before its end.

Preferably, the number of amplicons to be amplified in the process at the beginning of the process is less than 500,000, more preferably less than 200,000, most preferably less than 100,000, most preferably less than 10,000. By this embodiment of the invention can be advantageously prevented that the duplication reached saturation before its end.

In one embodiment of the invention, the nucleic acids are amplified by means of a polymerase chain reaction (PCR), in which a cycle consisting of the steps denaturation, annealing and elongation is repeated.

The cycle can be run so many times until the desired amount of duplication is achieved. The number of passes of the polymerase chain reaction cycle is preferably greater than 45, more preferably greater than 60, more preferably greater than 80, more preferably greater than 100, more preferably greater than 120, most preferably greater than 160, most preferably greater than 200. With a large number of passes advantageously a particularly high duplication can be achieved.

The number of cycles of the polymerase chain reaction cycle is preferably less than 1000, more preferably less than 750, most preferably less than 500. With a not too high number of passes of the cycle, advantageously the duration of duplication may be reduced. In addition, advantageously, the negative effects of contaminants or the consumption or damage of reactants, such as a polymerase used in the process can be kept low.

Preferably, the PCR uses primers conjugated nanoparticles. When carrying out the PCR, double-stranded PCR products thus preferably arise, wherein in each case at least one single strand of the double-stranded PCR products is conjugated to a nanoparticle. By excitation of the nanoparticles, it is advantageously achievable in this embodiment to generate the denaturation temperature around the nanoparticles and to perform the denaturation of the double-stranded PCR products without heating the entire reaction volume. This can accelerate the denaturing and thus the PCR run faster. In a further preferred embodiment, the annealing temperature and the elongation temperature are also generated by the excitation of the nanoparticles. Thus, preferably only a small amount of energy has to be transferred to the annealing and elongation temperature compared to heating the whole sample. Particularly preferably, denaturation, annealing and elongation of the PCR takes place without global heating, but exclusively via local heating by excitation of the nanoparticles. As a result, the method can be carried out without a means for global heating, so that less equipment is required to carry out the method.

In a further preferred embodiment, the method comprises a global heating step. In this case, the temperature of at least one process step is at least partially achieved by global heating. In a particularly preferred embodiment of the invention, the method is a PCR and the annealing temperature is achieved by globally heating the reaction volume. Most preferably, the reaction volume is maintained throughout the process by global heating within a predetermined temperature range in which annealing occurs. The elongation temperature and the denaturation temperature are achieved by excitation of the nanoparticles. Thus, advantageously, the device that generates the global heating can be kept very simple in construction because it only needs to hold a predetermined temperature.

In a further preferred embodiment, the annealing temperature and the elongation temperature are achieved by global heating and exclusively the denaturation is generated by excitation of the nanoparticles. So can be achieved with advantage that the device that causes the global heating, must produce a temperature cycle with only two different temperatures and thus structurally simple can be kept. The elongation and the annealing usually take place each in a narrow temperature range. On the other hand, only a certain temperature has to be exceeded for denaturing. Therefore, inhomogeneities in the excitation of the nanoparticles to produce the denaturation may be less of a problem than setting the annealing and elongation temperature. Consequently, a preferred embodiment, in which the excitation of the nanoparticles exclusively serves for denaturing, can be realized in a technically simpler manner. This applies in particular to the particularly preferred case in which the annealing temperature and the elongation temperature are very close together, e.g. B. at an annealing temperature of 60 ° C and a elongation temperature of 72 ° C, so that the global heating must produce only a small increase in temperature.

In a particularly preferred embodiment, the annealing temperature is equal to the elongation temperature. The procedure is carried out as a PCR. If the annealing temperature is equal to the elongation temperature, usually only one temperature cycle with two different temperatures is necessary to carry out the PCR, whereby the process can be carried out in a simple structure. Particularly preferably, the melting temperatures of the primer and the DNA polymerase used are chosen so that at the melting temperature, the DNA polymerase used can still synthesize DNA at a satisfactory rate. In a particularly preferred embodiment, the elongation temperature, which is equal to the annealing temperature, is achieved by global heating and the denaturation is achieved by excitation of the nanoparticles. In this way, the device that causes global heating can be made structurally simple because it only needs to maintain one temperature.

In a preferred embodiment, the excitation of only a portion of the nanoparticles takes place at any point in the process. These can z. For example, the means which serve to excite the nanoparticles can be designed such that they excite the nanoparticles present there only in a part of the reaction volume. In a particularly preferred embodiment, the nanoparticles are optically excited by a laser and the optics which directs the light of the laser into the reaction volume are designed so that light is conducted only in a part of the reaction volume. The fraction of nanoparticles that is excited preferably changes during the course of the process. In other words, a first amount of the nanoparticles that are excited at a first time is not identical to a second amount of the nanoparticles that are excited at a second time. In this case, any number of nanoparticles in the first and any number of nanoparticles in the second amount may be present as long as the first and second amounts are not identical. Of the two mentioned amounts can z. B. one with the other partially overlap, so that the two sets form an intersection. The one lot can z. B. may be a subset of the other amount, so that contains a lot less nanoparticles than the other amount. The two quantities can z. B. also be designed so that they do not form an intersection and thus no nanoparticles at the same time in the first amount and in the second amount is present. One of the two quantities can also be the empty amount, so that z. For example, nanoparticles can be excited at one time, and nanoparticles can not be excited at another time. In a preferred embodiment, the first and second amounts contain substantially the same number of nanoparticles. Particularly preferably, a laser excites different proportions of the nanoparticles at different times. As a result, in the embodiment of the method, a laser with a smaller power can be used which is just sufficient to excite a proportion of the nanoparticles. In a particularly preferred embodiment, two or more lasers are used to excite different portions of the nanoparticles. As a result, it is advantageously possible to excite different proportions of the nanoparticles without the need for an optical element which directs the laser to different parts of the reaction volume.

In a further preferred embodiment of the invention, a directed movement of the sample takes place relative to a stimulating field, so that at different times nanoparticles are excited in different partial volumes of the sample. Particularly preferably, the exciting field is the light of a laser. In a very particularly preferred embodiment, the light of the laser is directed by an optical element such that at different times nanoparticles in different subvolumes of the reaction volume are excited by the light. The optical element may be arranged to be movable, for. For example, the optical element may include a movable mirror, a spatial modulator or an acousto-optic modulator. It can also be arranged to be movable itself the laser. The movement of the sample can also be realized by moving the reaction vessel containing the sample. In a particularly preferred embodiment, both the laser beam and the reaction vessel is moved. In a further preferred embodiment, the sample is moved in the reaction volume, so that the light of the laser detects different partial volumes of the sample at different times. This can be z. B. be achieved in that the sample is mixed in the reaction volume, z. B. by a magnetic stirrer. The reaction volume can, for. B. assume an elongated shape, for. As a channel or a hose. The sample can z. B. are moved through a channel, wherein the sample passes through a laser beam at one or more locations. More preferably, a sample flows through a channel and passes n positions at which a laser beam is directed at the sample in the channel, whereby the linear flow of the sample through the n laser beams passes through n-cycle PCR. Thus, advantageously, the method can be carried out with a small number of moving parts. By using a channel, miniaturization, e.g. B. in the sense of a lab-on-a-chip possible. Preferably, by the laser beam denaturing is produced while elongation and annealing temperatures are generated by global heating. The elongation equal to the annealing temperature is particularly preferred, so that only one temperature must be maintained by global heating. In this way, the inventive method can be advantageously carried out with little effort.

In a preferred embodiment, the method uses a DNA polymerase which is thermolabile. In the case where the excitation of the nanoparticles is used for denaturation, it can be avoided that the entire reaction volume is exposed to high temperatures. Rather, it is possible to bring only the immediate environment of the nanoparticles to the denaturation temperature. The DNA polymerases that are not in this immediate environment are thus not exposed to high temperatures. This makes it possible to use DNA polymerases that are not heat stable, that are thermolabile. Thus, by including the thermolabile DNA polymerases, a greater variety of polymerases is available for the process of the present invention. The greater variety of DNA polymerases allows the reaction conditions to be altered to a greater extent while maintaining sufficient function of the particular DNA polymerase. In order that the nucleic acids to be amplified can bind to the negatively charged oligonucleotides on the nanoparticles, it may be necessary to use substances - in particular salts - in the sample in a concentration which adversely affect the function of a thermostable DNA polymerase, which increases the efficiency of the Method lowers. The greater variety of DNA polymerases - especially those that have a high tolerance for salts - can thus lead to an increase in the efficiency of the process is achieved. Part of the wider range of DNA polymerases are small DNA polymerases such. The Klenow fragment and Phi29. In the vicinity of the nanoparticles, large thermostable DNA polymerases can be sterically hindered by the applied and optionally already elongated primers. As a result, the DNA polymerase may not get to the nucleic acid to be copied or break off the DNA polymerase before synthesizing a complete copy of the original or complement, which means a reduction in the efficiency of the process. The greater choice of DNA polymerases thus allows an increase in the efficiency of the process. Due to the larger selection of DNA polymerases, enzymes with lower production costs are also advantageously available. The DNA polymerases that are not in the immediate vicinity of the nanoparticles undergo less heat-induced deactivation. As a result, a smaller amount of DNA polymerase can advantageously be used in the process.

In a preferred embodiment of the invention, both soluble primers and primers on nanoparticles are present in the reaction volume. The soluble primers are not conjugated to nanoparticles but are present dissolved in the sample. The soluble primers are preferably smaller in size than the nanoparticle-primer conjugates and can be in higher concentration than the nanoparticle-primer conjugates. Therefore, the soluble primers can provide better and faster access to long, double-stranded nucleic acids, such as. B. have genomic DNA. In a particularly preferred embodiment, in a first step of the method, the long, double-stranded nucleic acids are denatured by globally heating the entire reaction volume, whereupon the dissolved primers hybridize with the nucleic acids. The PCR initially takes place in one or more cycles of global heating, while the DNA polymerase synthesizes the desired, short copies of the long, double-stranded nucleic acids. Thereafter, the PCR is continued, with local heating by excitation of the nanoparticles is also used.

Brief description of the figures

Show it:

1 in a schematic representation, the nanoparticles according to the invention, which are conjugated with filling molecules, spacer sequences, abasic modifications and primer sequences;

2 in a further schematic representation, the nanoparticles according to the invention which are conjugated with filling molecules, spacer sequences, abasic modifications and primer sequences;

3 in a schematic representation of a structure for carrying out the method according to the invention with a laser, a two-dimensional mirror scanner and a sample;

4 in a schematic representation of a further structure for carrying out the method according to the invention with a laser, a mirror and a relative to the laser beam moving sample;

5 in a schematic representation of another structure for carrying out the method according to the invention with a laser, a one-dimensional mirror scanner and a one-dimensional moving sample;

6 in a schematic representation, the nanoparticles according to the invention and the test probes according to the invention for the positive detection of DNA;

7 in a schematic representation of another structure for carrying out the method according to the invention with a laser, a two-dimensional mirror scanner and sample tube in a water bath;

8th in two diagrams the results of amplification reactions with global and local heating with test probes for the positive detection of DNA;

9 a schematic representation of the nanoparticles according to the invention and the test probes according to the invention with terminating modifications for the negative detection of DNA;

10 in a schematic representation of another structure for carrying out the method according to the invention with a laser, two heating blocks with recesses, multiwell plate and film and lens and photodiode;

11 in a diagram, the results of amplification reactions with test probes for the negative detection of DNA; and

12 in a schematic representation of a first laser for exciting nanoparticles in a sample tube and a second laser and a photodiode for measuring the transmission of the sample.

Detailed description of the invention with reference to several embodiments

1 shows an embodiment of the method according to the invention for the amplification of nucleic acids 1 which is designed as a PCR. In a reaction volume 2 are first nanoparticles 3 contain. The first nanoparticles 3 have on their surface oligonucleotides 4 on, like in 1a shown. A variety of oligonucleotides 4 each contain a primer sequence as a partial sequence 5 with the sequence A and as a further, optional partial sequence a spacer sequence 6 S and an optional abasic modification 7 between primer sequence 5 A and spacer sequence 6 S. The spacer sequence 6 S serves to the primer sequence 5 far enough from the surface of the nanoparticles 9 Keep away so that a nucleic acid to be amplified 1 with a better efficiency to the primer sequence 5 can bind and the DNA polymerase 11 better access to the primer sequence 5 Can be found. The abasic modification 7 prevents overwriting of the spacer sequence by the polymerase 11 , The oligonucleotides 4 with the primer sequence 5 A are z. With a thiol bond on the surface of the first nanoparticles 3 attached, leaving the 3'-end of the first nanoparticle 3 turned away. Optionally, another variety of oligonucleotides may be present 4 on the surface of the first nanoparticles 3 are, these are the filling molecules 10 F. With the filling molecules 10 can the charge of the nanoparticles 9 be modulated so that it does not cause unwanted aggregation of the nanoparticles 9 comes. In addition, the filling molecules 10 the distance of the primer sequences 5 to each other on the surface of the nanoparticles 9 enlarge so that the nucleic acids to be amplified 1 and the DNA polymerase 11 better access to the primer sequences 5 to have. This can increase the efficiency of the process. The spacer sequence 6 is preferably at least as long as the filling molecules 10 so that advantageously the primer sequences 5 from the filling molecules 10 protrude.

In the reaction volume 2 there is a sample 12 containing the first nanoparticles 3 out 1a with the primer sequences 5 , Spacer sequences 6 , Abasian modification 7 and filling molecules 10 contains and in addition dNTPs and DNA polymerase 11 has, in addition, further, necessary for a PCR reagents. A nucleic acid to be detected 1 can in the sample 12 to be available. In this embodiment, the nucleic acid to be detected is 1 a single-stranded DNA, also called the original 13 is designated and has a partial sequence A 'and a partial sequence B'. The original 13 may also have other subsequences, z. B. as overhangs at the 5 'or 3' end or between the two partial sequences A 'and B'. In 1b binds the original 13 with its partial sequence A 'to the primer sequence 5 A on the surface of the first nanoparticles 3 , In 1c is shown to be a DNA polymerase 11 to the original 13 and those with the original 13 hybridized primer sequence 5 A binds. Thereupon synthesizes the DNA polymerase 11 in one Elongation step in 1d is shown starting from the 3 'end of the primer sequence 5 A to the original 13 complementary nucleic acid 1 that as complement 14 is called and with the spacer sequence 6 on the surface of the first nanoparticle 3 connected is. In 1e becomes the first nanoparticle 3 then irradiated with light from the first nanoparticle 3 due to its plasmonic or material properties is absorbed and converted into heat. The heat is transferred to the environment of the first nanoparticle 3 and is in the range of the original 13 and the hybridized, newly synthesized complement 14 sufficient for that the original 13 from the complement 14 denatured. The original 13 is now free again, as in 1f so it attaches to another primer sequence 5 can bind and other nanoparticle-bound complements 14 can be synthesized in further cycles of the process. This results in a linear increase in the concentration of the complements 14 with increasing number of cycles.

In one embodiment of the method, after extending the primer sequence 5 on the surface of the first nanoparticles 3 in which a nanoparticle-bound complement 14 arises, a free reverse primer 16 used, which binds to the 3 'end of the complement. In 1g is shown to be the already synthesized complement 14 with the partial sequences A and B, via a spacer sequence 6 and an abasic modification 7 on the surface of the first nanoparticle 3 is connected with a previously free in the sample 12 existing primer 8th B 'hybridizes. It has the primer 8th the sequence B 'and is with the partial sequence B of the complement 14 connected. Starting from the primer 8th with sequence B ', the DNA polymerase synthesizes a copy of the original 13 , In 1g it is also shown that the original 13 to another primer sequence 5 A on the surface of the first nanoparticle 3 has bound and a DNA polymerase 11 starting from the primer sequence 5 A another complement 14 synthesized. The original 13 , the copy of the original 13 and the two complements associated with the first nanoparticle 14 are in 1h shown. Subsequent denaturation by excitation of the first nanoparticles 3 causes the original 13 and release his copy. It can both the original 13 as well as his copy in subsequent steps of the procedure as a template for duplication serve. After a waiting period, if necessary for hybridization of original 13 and copies of the original 13 with primer sequences 5 A on the first nanoparticles 3 and of free primers 8th B 'with on the first nanoparticles 3 already elongated primer sequences 5 necessary, may be with a further excitation of the first nanoparticles 3 the next cycle of the process will be performed. Preferably, the cycle is repeated until there are enough extended primer sequences 5 on the first nanoparticles 3 and / or enough copies of the original 13 in the sample 12 proof of the duplication or the presence of the original 13 in the sample 12 to carry out. Through a free primer 8th B 'is as in 1g and 1h shown an exponential duplication of the original 13 possible. In 1a to 1f is without this free primer 8th but only a linear amplification of the nanoparticle-bound complement 14 to reach.

In 2 an embodiment of the method according to the invention is shown, in which nanoparticles 9 in a rehearsal 12 are located. The nanoparticles 9 have filling molecules on their surface 10 F on. Further, the nanoparticles 9 with oligonucleotides 4 conjugated. A first sort of oligonucleotides 4 consist of a spacer sequence 6 S and a primer sequence 5 A and an optional abasic modification 7 between primer sequence 5 A and spacer sequence 6 S. A second variety of oligonucleotides 4 consists of a spacer sequence 6 S and a primer sequence 5 B 'and an optional abasic modification 7 between primer sequence 5 B and spacer sequence 6 S. The original to be duplicated 13 in this embodiment is a single-stranded DNA molecule having partial sequences A, C and B (not shown). By a DNA polymerase 11 became one to the original 13 complementary strand starting from the primer sequence B 'on the surface of the nanoparticle 9 synthesized, so that, as in 2a shown now on the nanoparticle 9 a single strand of DNA having the sequence S, B ', C' and A 'is located. At the same time is in 2a to see that by a DNA polymerase a copy of the original 13 starting from the primer sequence 5 A, with the spacer sequence 6 S and the optional abasic modification 7 on the surface of the nanoparticle 9 is synthesized. As indicated by an arrow in 2a shown hybridizes on the nanoparticle 9 attached copy of the original 13 with its partial sequence B to a primer sequence 5 B 'on the surface of the same nanoparticle 9 , By a second arrow in 2a This is shown to be on the surface of the nanoparticle 9 synthesized complement 14 with its partial sequence A 'to a primer sequence 5 A on the surface of the same nanoparticle 9 hybridized. The result of the two mentioned hybridizations is in 2 B shown. In doing so form both the original 13 as well as the complement 14 a loop on the surface of the nanoparticle 9 , In 2c can be seen that starting from the primer 8th B 'one to the original 13 Complementary strand is synthesized via a spacer sequence 6 S with the surface of the nanoparticle 9 connected is. By another DNA polymerase 11 is starting from the primer sequence 5 A is a copy of the original 13 synthesized, which also has a spacer sequence 6 with the surface of the nanoparticle 9 connected is. The synthesis of the polymerase 11 ends at the abasic modification 7 , The result of the two syntheses is in 2d to see. In this embodiment, both the forward primer are located 15 as well as the reverse primer 16 on the same nanoparticle 9 , In this way, a newly synthesized strand of DNA can be returned to a primer 8th on the same nanoparticle 9 hybridize. This can lead to an acceleration of the method according to the invention since the newly synthesized DNA strand does not have to travel far to access a complementary primer 8th hold true. Rather, the newly synthesized DNA strand can be particularly fast to a complementary primer 8th on the surface of the same nanoparticle 9 which is particularly facilitated by the local concentration of primers 8th on the nanoparticle 9 is particularly high. After exciting the nanoparticle 9 in 2d , for example by a laser 17 , dehybridize the copies of the original 13 and the copies of the complement 14 , each using spacer sequences 6 optional abasic modification 7 on the surface of the nanoparticle 9 are attached. Thereupon can be a copy of the original 13 attached to a nanoparticle 9 attached, is with a complement 14 that is on the surface of another, identical nanoparticle 9 attached, hybridize. Hybridization turns the nanoparticles 9 connected, so that a measurable change arises. The measurable change may be, for example, in a color change of the sample 12 consist. By the in 2a to 2e illustrated embodiment of the method according to the invention, it is possible to provide a simple test, the proof of the original 13 serves.

In 3 a structure is shown which is suitable for carrying out the method according to the invention. The structure contains a light source 18 which in this example is called a laser 17 is executed and a two-dimensional mirror scanner 19 , the light from the laser 17 to the test 12 can direct. The two-dimensional mirror scanner 19 can deflect the laser beam in two dimensions. The denaturation in the sample 12 happens in this structure in that a laser beam on a part of the sample 12 is focused. During the process, the laser beam is deflected so that it hits different parts of the sample 12 meets. In the in 3 As shown, the laser beam passes through the mirror scanner 19 so distracted that the laser beam is the reaction volume 2 in which the sample is 12 is located, descends lines. The path that the laser beam travels in 3 in the sample 12 shown in dashed lines. This means that at any point in the procedure only parts of the sample 12 can be excited, can laser 17 be used with a smaller power. Since excitations of under one microsecond are sufficient to DNA using optothermisch heated nanoparticles 9 denature, can at typical focus diameters of a laser 17 from about 10 to 100 microns, a laser beam at a speed of about 10 to 100 m / s, the sample 12 and thereby denature the DNA at each point crossed by the laser beam. This allows a very fast scanning even of large sample volumes. The complete scanning of an area of 1 cm ² takes z. B. at a focus diameter of 78 microns and 128 lines in the line spacing of 78 microns and a line length of 1 cm at a speed of the scanning laser beam of 10 m / s only 128 ms. Does the volume z. B. a depth of 10 mm, so a volume of 1 ml can be processed (of course, this must, inter alia, ensure that the intensity of the stimulation over the entire depth is sufficiently high). This is advantageously much shorter than would normally require a denaturation step by global heating. With optical elements such. B. a in 3 shown mirror scanner 19 , which may be implemented as a galvanometric scanner, and so-called F theta lenses can provide good homogeneity of focus quality and size over the entire sample being scanned 12 be achieved. Alternatively to a continuously emitting laser 17 can also be a pulsed laser 17 or a thermal radiator can be used.

In 4 a structure for carrying out the method according to the invention is shown, in which a laser 17 , and a mirror 20 is immovably arranged and the laser beam of the laser 17 through the mirror 20 to the test 12 is steered. Here is the sample 12 movably arranged in two dimensions, allowing movement of the sample 12 , the whole rehearsal 12 or large parts of the sample 12 from the focus of the laser 17 can be detected.

In 5 a structure for carrying out the method according to the invention is shown, in which a laser 17 is immovably arranged and a mirror scanner 19 the laser beam of the laser 17 can distract in one direction. The sample 12 is arranged to be movable in one direction, so that by movement of the mirror scanner 19 and the sample 12 the whole sample 12 or large parts of the sample 12 can be detected by the laser beam.

One way to detect a nucleic acid 1 by PCR according to the invention is in 6 shown. The first nanoparticles are located in a sample 3 that have filling molecules on their surface 10F and first oligonucleotides 21 exhibit. The first oligonucleotides 21 consist of a spacer sequence 6S and a primer sequence 5 A, as in 6a shown. If an original 13 with the partial sequences A 'and B' in the sample 12 is present, the original hybridizes 13 on the complementary primer sequence 5 A on one the first nanoparticle 3 , By a DNA polymerase 11 is starting from the primer sequence 5 A is the complement 14 written with the subsequences A and B, so the complement 14 via the spacer sequence 6S with the surface of the first nanoparticle 3 is connected, as in 6c shown. In a next step, the in 6d shown test probes 22 given to the sample. The test probes 22 are second nanoparticles 23 that have filling molecules on their surface 10 and second oligonucleotides. The second oligonucleotides contain a spacer sequence 6S and a test sequence 5B '. The test sequence 5B 'can with the complementary partial sequence B of the complement 14 on the surface of the first nanoparticle 3 hybridize, as in 6f shown. This will be the first nanoparticles 3 and second nanoparticles 23 connected, so that a measurable change can occur. If the original 13 in the sample 12 is absent, then arises on the surface of the first nanoparticles 3 no complement 14 , as in 6b you can see. As reflected on the first nanoparticles 3 but no complement 14 can, first nanoparticles 3 and second nanoparticles 23 do not connect and the measurable change does not arise. The sequence B 'in this embodiment is complementary to the sequence B, but it may also be complementary to parts of the sequence A. The spacer sequence 6S on the first nanoparticles 3 is with the spacer sequence 6S on the second nanoparticles 23 identical. In a further embodiment, however, on the first nanoparticles 3 and second nanoparticles 23 also different spacer sequences 6 be used. There may also be several different spacer sequences 6 be used on the same sort of nanoparticles. The buffering and hybridization conditions, e.g. As temperature, salt concentrations, nanoparticle concentrations, concentrations of other buffer additives, pH, are preferably chosen so that only after the extension of the primer sequence 5 A on the first nanoparticles 3 a hybridization can occur that involves the first nanoparticles 3 with the second nanoparticles 23 combines. The connection of the first nanoparticles 3 with the second nanoparticles 23 can z. B. be detected as redshift and broadening of the plasmon resonance in the extinction spectrum. The connection can also z. Example by measuring the change in transmission at one or more wavelengths after opto-thermal excitation of the nanoparticles and resulting denaturation of the nucleic acids 1 containing the first nanoparticles 3 with the second nanoparticles 23 connect, be detected. The test probes 22 can be provided in a specific hybridization buffer to which at least a portion of the sample 12 containing the first nanoparticles 3 contains, after the process step, the synthesis of the complement 14 allows, is added. The test probes 22 may also be present from the beginning of the procedure in the sample along with the first nanoparticles 3 available. In this case, the test probes 22 be passivated, so they are not considered primers 8th Act. The passivation of the test probes 22 may be that the primer sequence 5 on the test probes 22 is chosen so that at the annealing temperature during the PCR no hybridization of said primer sequence 5 with the original 13 takes place, but only after subsequent lowering of the temperature. The passivation of the test probes 22 can also be done so that the second oligonucleotides, the partial sequences of the original 13 contained at the 3 'end on the second nanoparticles 23 are attached, leaving the DNA polymerase 11 the second oligonucleotides can not extend. In this case, the second oligonucleotides may be free at their 5 'end or with the second nanoparticles 23 be connected. The test probes 22 may also be passivated by a base modification, e.g. B. with dideoxycytosine (ddC) at the 3 'end of the second oligonucleotides prevents the elongation.

In the execution of the procedure, which in 6 shown are first nanoparticles 3 of gold with a diameter of 60 nm with oligonucleotides 4 functionalized (after J. Hurst et al., Anal. Chem., 78 (24), 8313-8318, 2006 the content of which is incorporated herein by reference). In this case, part of an oligonucleotide 4 ID1 and as a filling molecule 10 to four parts oligonucleotide 4 ID2 used. After functionalization and 6 washes, the first nanoparticles are located 3 at a concentration of 200 pM in a PBS buffer (20 mM PBS, 10 mM NaCl, 0.01% Tween 20, 0.01% azide, 1 mM EDTA, pH 7.5). The amplification reaction is carried out in a total volume of 10 μl in 200 μl sample tubes 24 5 μl of DreamTaq PCR Master Mix 2x (purchased from Fermentas), 0.1 μl of NaCl 5 M, 0.1 μl of MgCl ₂ 250 mM, 0.1 μl of MgSO ₄ 250 mM, 1 μl of the functionalized first nanoparticles 200 μM, 1 μl oligonucleotide 4 ID3 (as original to be reproduced 13, while the concentration of the original to be determined is 13 in the total volume of 10 .mu.l z. 0 pM, 10 pM, 20 pM or 50 pM) dissolved in water with 100 nM oligonucleotide 4 ID4 (oligonucleotide 4 ID4 serves for the saturation of surfaces, eg. During storage of the original 13 before the reaction), 2.7 μl of water).

As in 7 shown, the sample tubes 24 in a glass cuvette 25 in a water bath 26 to a temperature of 65 ° C, which represents both the annealing and the elongation temperature brought. The water bath 26 In addition to the temperature control, the better coupling of the laser is also used 17 into the non-planar surface of the sample tubes 24 , The water in a water bath 26 allows the refractive index difference between the outside and the inside of the sample tubes filled with PCR reaction mix 24 is reduced and thus a refraction of the laser beam and thus a negative influence the focus quality and sharpness is suppressed. As a result, the coupling of the laser is advantageous 17 improved. The laser 17 which serves to excite the nanoparticles, is a frequency doubled diode pumped Nd: YAg laser (Coherent Verdi V10), which at an output power of 1.5 W with an F-theta lens (Jenoptik, focal length 100 mm) behind a mirror scanner 19 (Cambridge Technologies, Pro Series 1) into the sample tubes 24 in a water bath 26 is focused (focus diameter about 20 microns). The mirror scanner 19 allows the focus line by line through the sample tubes 24 to move, as already in 3 shown, and thus the entire PCR reaction volume to participate in the optothermal amplification. Per sample tube 24 become 400 lines with a distance of about 12 microns at a line speed in the sample tube 24 from about 2 m / s with the focus. This corresponds to one cycle in the first sample tube 24 , Subsequently, all other sample tubes are successively 24 trailed off, leaving each sample tube 24 has experienced a cycle. After waiting for 40 seconds after passing through the first sample tube 24 The next cycle is started and this is repeated until each sample tube 24 a total of 25 cycles has passed. As initial concentration of the original 13 will be in the first sample tube 24 0 pM, in the second sample tube 24 20 pM and in the third sample tube 24 50 pM chosen. The negative control is a fourth sample tube 24 in the water bath 26 used, which is also the original 13 in a concentration of 50 pM, but is not hit by the laser beam. After the first, second and third sample tubes 24 Has passed through 25 cycles, all four sample tubes 24 from the water bath 26 away. To study the effect of the laser cycles and the concentration of the original 13 serves a test probe 22 under the chosen buffer and hybridization conditions only to those by extension of the nanoparticle-bound primer 8th can hybridize resulting partial sequences. Here is the extension of the primer 8th complementary to the original 13 , as in 6c shown. For the production of test probes 22 become second nanoparticles 23 of gold with a diameter of 60 nm with oligonucleotides 4 functionalized (after J. Hurst, supra ). It becomes part of an oligonucleotide 4 ID5 and as a filling molecule 10 to four parts oligonucleotide 4 ID2 used. After functionalization and 6 washes, the second nanoparticles are located 23 at a concentration of 200 pM in a PBS buffer (20 mM PBS, 10 mM NaCl, 0.01% Tween 20, 0.01% azide, 1 mM EDTA, pH 7.5). For the hybridization of the oligonucleotides 4 on the first nanoparticles 3 with the oligonucleotides 4 on the second nanoparticles 23 a modified phosphate buffer is used (13 mM PBS, 200 mM NaCl, 0.02% Tween 20, 1 mM EDTA, 20 mM sodium citrate, 1 μg / ml PVP10, pH 7.5). 10 μl of hybridization mixture contains 2.25 μl of the modified phosphate buffer, 3 μl of formamide, 2 μl of NaCl 5M, 0.25 μl of the 200 pM test probe solution and 2.5 μl of the corresponding PCR solution from the optothermal amplification containing the first nanoparticles 3 contains. If there is enough of the original in the sample tube 13 with the sequence ID3 present, it is on the surface of the first nanoparticles 3 the oligonucleotide 4 extended with the sequence ID1 and can with the oligonucleotide 4 with the sequence ID5 on the surface of the test probe 22 hybridize, as in 6f shown. The proof of this hybridization is carried out by means of optothermal excitation of the nanoparticles (according to EP 2162549 the content of which is incorporated herein by reference). The sample tubes 24 become like in 12 shown with pulses of a first laser 28 Shotged (50 μs pulse duration, 532 nm wavelength, about 700 mW peak power, focus diameter about 30 microns). As a result, the nanoparticles are heated optothermally and give off heat to their environment. If first nanoparticles 3 and second nanoparticles 23 by the hybridization of oligonucleotides 4 , as in 6f are connected, they are separated by the laser pulse. This can be done by a in 12 illustrated second laser 29 (Wavelength here 630 nm, power 5 mW continuously), its focus (30 μm diameter) with the focus of the first laser 28 , which is preferably used exclusively for dehybridization is superimposed and the extinction before and after the laser pulse of the first laser 28 queries. The optical path on which the change in absorbance is optothermally induced and measured is approximately 2 mm. The intensity of the light transmitted through this layer of the second laser 29 is using a photodiode 30 measured. From the difference of the photodiode current before and after the pulse, the optothermally induced transmission change is determined, which is produced by dehybridizing the extended first oligonucleotides and second oligonucleotides between the nanoparticles and the subsequent diffusing out of the nanoparticles.

In 8a the relative transmission change is shown by the laser pulse of the first laser 28 and the resulting dehybridization of the oligonucleotides 4 between the first nanoparticles 3 and second nanoparticles 23 and measure the presence of gold-DNA-gold bonds in the sample tube 24 is. Below the diagram in 8a the number of cycles passed can be seen in a first line. In a second line, which is below the first line, is the concentration of the original 13 in pM in the sample tube 24 presented before the duplication. Shown are on the right side of the diagram in 8a in section B from left to right the first, second and third sample tubes 24 , respectively 25 has undergone optothermal cycles as well as the fourth sample tube 24 without optothermal treatment. Here you can clearly see that the measured Transmittance change as an indicator of gold-DNA-gold bonds with increasing concentration of the original 13 before duplication increases when the 25 cycles have been completed. For the first sample tube 24 without original 13 and the fourth sample tube 24 Without optothermal treatment, only a small change in transmission is observed. This shows that here no extension of the primer sequences 5 on the first nanoparticles 3 took place and thus no binding to the test probe 22 is possible. Only after passing through the optothermal cycles and in the presence of the original 13 it may lead to an extension of the primer sequences 5 on the first nanoparticles 3 through the DNA polymerase 11 come, resulting in a compound of the first nanoparticles 3 with the second nanoparticles 23 and finally leads to a change in transmission as a result of the optothermically induced separation of the nanoparticles.

As comparison is in 8a in section A on the left, we can see the result of an analogous experiment, in which the heating of the DNA is not localized by optothermal excitation of the nanoparticles 9 but globally for the entire reaction volume 2 in a conventional thermocycler (Labnet Multi Gene II) takes place. Here, from left to right, the first to fourth sample tubes 24 whose contents are identical to the experiment described in the previous paragraph. First, second and third sample tubes 24 were subjected to a classical PCR protocol (93 ° C for 1 s, 53 ° C for 20 s, 35 cycles). As in the case of optothermal heating, it can also be seen that the more original 13 before duplication in the respective sample tube 24 was present, the greater the measured change in transmission due to the laser pulse and the thereby dehybridisierende DNA between the first nanoparticles 3 and second nanoparticles 23 and is the measure of the presence of gold-DNA-gold bonds in the solution. The fourth sample tube 24 that is 50 pM of the original 13 contains, but was not heated cyclically, shows almost no change in transmission. Thus, not enough primer sequences were used here 5 on the first nanoparticles 3 extended.

8b shows a similar experiment with global heating of the total reaction volume 2 but at a constant concentration of the original 13 in the sample tube 24 of 10 pM before duplication (second row below the chart) and increasing number of cycles (first row below the chart). Here it can clearly be seen that as the number of cycles increases, so does the measured change in transmission, which is a clear sign that there are more primers on the first nanoparticles 3 be extended as more cycles are passed and thus a clear indication that the origin of the signal being measured is actually the elongation of the oligonucleotides 4 on the first nanoparticles 3 through the DNA polymerase 11 is.

Another way to prove duplication is in 9 shown. 9a and 9c summarize the exponential amplification using a resolved reverse primer 16 B 'as already described in 1a to 1h is shown. Additionally are in the sample 12 test probes 22 , The test probes 22 consist in this embodiment of second nanoparticles 23 on their surface next to optional filling molecules 10F also with the test sequence 31A 'are functionalized. Optionally, between the test sequence 31A 'and the surface of the second nanoparticles 23 another spacer sequence 6S which are not identical to the spacer sequence 6S on the first nanoparticles 3 out 1 or 9a must be and an optional abasic modification 7 between test sequence A and spacer sequence 6S , The test sequence A 'is at least part of the primer sequence 5 A on the first nanoparticles 3 complementary. The test sequence A 'occurs over the primer sequence 5 A in competition with those in the process in 1a to 1h formed copies of the original 13 with the partial sequence A '. That is, if many copies of the original 13 are present, then are the primer sequences 5 A on the surface of the first nanoparticles 3 already with the partial sequences A 'of the copies of the original 13 occupied. The primer sequences 5 A can not or only to a small extent with the test sequences A 'on the second nanoparticles 23 hybridize. This will be the first nanoparticles 3 with the second nanoparticles 23 not or only to a limited extent. As in 9c are the elongated primer sequences 5 A on the first nanoparticles 3 hybridizes with the original 13 and its copies, thereby forming rigid, double-stranded DNA that can present a steric barrier, thereby also joining first nanoparticles 3 and second nanoparticles 23 with a high number of copies of the original 13 prevented. In the absence or small number of original 13 and copies of the original 13 , lie the first nanoparticles 3 predominantly with unoccupied primer sequences 5 A before, as in 9b shown. Now the test sequences 31 the test probes 22 with the unoccupied primer sequences 5 A on the first nanoparticles 3 hybridize. In the process, the first with the second nanoparticles 23 connected, as in 9b shown. In this embodiment, the extent of the compound of first nanoparticles 3 and second nanoparticles 23 the weaker the more copies of the original 13 caused by the duplication reaction, which in turn depends on the concentration of the original 13 at the beginning of the amplification reaction. The buffering and hybridization conditions (eg temperature, salt concentrations, nanoparticle concentrations and sizes, concentrations of other buffer additives, pH) are chosen so that when specific extension of the primer sequence 5 A and completed synthesis of copies of the original 13 the most efficient possible suppression of the hybridization of the primer sequences 5 A with the test sequences 31 A 'takes place. At the same time, the conditions mentioned are selected such that, if duplication does not take place, the most efficient possible hybridization of the primer sequences 5 A with the test sequences 31A 'arises. The hybridization resulting compound of first nanoparticles 3 with second nanoparticles 23 can z. B. be detected as redshift and broadening of the plasmon resonance in the extinction spectrum or by measuring the transmission change at one or more wavelengths after opto-thermal excitation of the nanoparticles and resulting denaturation of the nanoparticle-connecting DNA. Alternatively, the detection or quantification of the copies of the original produced in the process 13 z. B. by PCR, real-time PCR, quantitative real-time PCR, gel electrophoresis or by means of dye-labeled probes. The test probes 22 can be passivated, so they are not considered primers 8th Act. The passivation of the test probes 22 may be that the test sequence 31 on the test probes 22 is chosen so that at the annealing temperature during the PCR no hybridization of said test sequence 31 with the original 13 takes place, but only after subsequent lowering of the temperature. The passivation of the test probes 22 can also be done so that the test sequences 31 at the 3 'end on the second nanoparticles 22 are attached, leaving the DNA polymerase 11 can not extend the test sequences. In this case, the test sequences 31 be free at its 5 'end or with the second nanoparticles 23 be connected. The test probes 22 may also be passivated by a base modification, e.g. With dideoxycytosine (ddC) at the 3 'end of the test sequences 31 Elongation prevented. The test probes 22 can also only after completion of the optothermal amplification reaction to the sample 12 be added.

For the in 9 illustrated embodiment of the method become first nanoparticles 3 of gold with a diameter of 60 nm with oligonucleotides 4 functionalized, as in the in 6 shown embodiment, but now with oligonucleotides ID6. After functionalization and 6 washes, the first nanoparticles are located 3 at a concentration of 200 pM in a PBS buffer (5 mM PBS, 10 mM NaCl, 0.01% Tween 20, pH 7.5). For the production of test probes 22 become second nanoparticles 23 from gold with a diameter of 10 nm with oligonucleotides 4 ID7 functionalized (after J. Hurst, supra ) terminated with dideoxycytosine (ddC) at the 3'-end. After functionalization and 6 washes, the second nanoparticles are located 23 at a concentration of 8 nM in a PBS buffer (5 mM PBS, 10 mM NaCl, 0.01% Tween 20, pH 7.5). The amplification reaction is carried out in a total volume of 20 μl in a multiwell plate 32 with transparent bottom (supplied by Greiner Bio One) (4 μl Apta Taq Master Mix 5x with MgCl 2 (purchased from Roche), 2 μl NaCl 450 mM, 2 μl MgCl 2 90 mM, 2 μl Tween 20 1%, 2 μl tetramethylammonium chloride, 50 mM, 2 μl of water, 2 μl of the functionalized first nanoparticles 200 pM, 1 μl of the functionalized second nanoparticles 8 nM, 1 μl of oligonucleotide 4 ID8 as dissolved reverse primer and 2 .mu.l of target sequence as original to be reproduced 13. It is in the total volume z. B. 2E7, 2E6, 2E5, 2E4, 2E3, 2E2 copies of genomic DNA of the bacterium E. coli (Escherischia coli), the partial sequence of the original 13 contains in the sample 12, or 2E7 copies of genomic DNA of the bacterium MRSA (methicillin-resistant Staphylococcus aureus), which is the original 13 not included in the sample 12 , The genomic DNA of Escherischia coli and MRSA was previously extracted with a commercial extraction kit (Qiagen DNeasy Blood & Tissue Kit) from E. coli bacteria or MRSA bacteria. The genomic DNA thus recovered was frozen for storage and heat treated at 99 ° C for 10 minutes prior to insertion into the amplification reaction.

As in 10 shown, the multiwell plate 32 with a transparent foil 33 (supplied by Carl Roth) closed. The multiwell plate 32 is from below with a first heating block 34 associated, the recesses 35 contains such that the laser 17 unhindered to reach the transparent bottom of the multiwell plate. By tempering the first heating block 34 the global sample temperature can be set, representing both annealing and elongation temperatures. The foil 33 comes with a second heating block 36 associated, which also has recesses 35 contains such that the laser can propagate unhindered after passing through the sample. The second heating block 36 It should prevent part of the sample on the film 33 can condense. The temperature of the second heating block 36 is preferably selected to be at least as high as that of the first heating block 34 , more preferably at least 5 ° C higher and most preferably at least 10 ° C higher than that of the first heating block. In the present exemplary embodiment, the temperature of the first heating block is 62 ° C., that of the second heating block is 80 ° C. Under these conditions there is no hybridization between the test sequence 24 and primer sequence 5 instead, so are first nanoparticles 3 and second nanoparticles 23 not connected to each other and the primer sequence 5 is not blocked for the duplication reaction. At lower temperatures, the z. B. prevails during the mixing of the reaction components, the first nanoparticles 23 and the primer sequences 5 partially or completely through the test sequences 31 and the test probes 22 blocked and thus protected against non-specific binding and hybridization. This can be used to advantage for the specificity of the amplification reaction. The laser 17 which serves to excite the nanoparticles, is a frequency-doubled diode-pumped Nd: YAg laser (Excel, Laser-Quantum), which widens at a power output of 1.8 W by means of a telescope approximately 3-fold and then with a telecentric F-theta Lens (Qioptiq, focal length 100 mm) behind a mirror scanner 19 (Cambridge Technologies, Pro Series 1) into the multiwell plate 32 is focused (focus diameter about 20 microns). The mirror scanner 19 allows the focus line by line through the multiwell plate 32 to move, as already in 3 shown, and thus the entire PCR reaction volume to participate in the optothermal amplification. Per well, 250 lines are traversed with a distance of about 15 μm at a line speed in the well of about 8 m / s with the focus. This corresponds to one cycle in the first well. Subsequently, all other wells are sequentially driven, so that each well has undergone a cycle. After a waiting period of 3 s after passing through the first well, the next cycle is started and this is repeated until each well has undergone a total of 210 cycles.

6 wells are examined which are in 11 are shown from left to right. The first well contains as control in the sample 2E7 copies of genomic DNA of the bacterium MRSA (Methicillin-resistant Staphylococcus aureus) containing the original 13 does not contain. In the second to sixth wells are in the sample 2E7, 2E6, 2E5, 2E4, 2E3, and 2E2 copies of genomic DNA of the bacterium E. coli, the partial as the original 13 contains, before. After all seven wells have each undergone 210 cycles and the opto-thermal amplification reaction is completed, the temperature of the first heating block becomes 34 reduced to 52 ° C, that of the second heating block 36 at 62 ° C. Under these conditions, a hybridization between test sequence 24 and primer sequence 5 take place if no or only a few originals 13 were present in the sample and thus the primer sequences 5 not or only to a limited extent by copies of the original 13 be blocked. In this case, first nanoparticles 3 and second nanoparticles 23 interconnected, as in 9b shown. If many copies of the original 13 are present, then are the primer sequences 5 A on the surface of the first nanoparticles 3 already with the partial sequences A 'of the copies of the original 13 occupied. The primer sequences 5 A can not or only to a small extent with the test sequences A 'on the second nanoparticles 23 hybridize. This will be the first nanoparticles 3 with the second nanoparticles 23 not or only to a limited extent. As in 9c are the elongated primer sequences 5 A on the first nanoparticles 3 hybridizes with the original 13 and its copies, thereby forming rigid, double-stranded DNA that can present a steric barrier, thereby also joining first nanoparticles 3 and second nanoparticles 23 with a high number of copies of the original 13 prevented. The proof of the hybridization takes place by means of optothermal excitation of the nanoparticles (according to EP 2162549 the content of which is incorporated herein by reference). The wells are going to, as in 10 shown with pulses of a laser 17 shot (50 μs pulse duration, 532 nm wavelength, about 1 W peak power, focus diameter about 20 μm). As a result, the nanoparticles are heated optothermally and give off heat to their environment. If first nanoparticles 3 and second nanoparticles 23 by the hybridization of oligonucleotides, as in 9b are connected, they are separated by the laser pulse. This can be proved by the fact that the laser 17 at low power (about 50 mW continuous) the extinction before and after the intense laser pulse of the laser 17 queries. The optical path on which the change in absorbance is optothermally induced and measured is about 1 mm. The low power during the absorbance measurement prevents the dehybridization of the primer sequence 5 and the test sequence 31 , which should only be induced by the intense laser pulse. The use of only one laser both for extinction measurement and for optothermal dehybridization preferably saves costs and an overlap of two laser foci. The intensity of the light transmitted through this layer of the laser 17 is using a photodiode 30 measured after the transmitted light of the laser 17 on a diffuser 27 is scattered. In this case, the lens reaches that part of the light that has been transmitted through the different wells always falls on the detector. From the difference of the photodiode current before and after the pulse, the optothermically induced transmission change is determined by the dehybridization of the primer sequence 5 and the test sequence 24 between the nanoparticles 8th and the subsequent diffusion of the nanoparticles 8th is produced. The result of the optothermally induced change in transmission is 11 shown. The first bar shows the measurement of a well with genomic DNA of the bacterium MRSA containing the original 13 not included as a partial sequence in the sample. Here, the primer sequences are not occupied by copies of the original, thus connecting first nanoparticles 3 and second nanoparticles 23 with each other and a significant change in transmission is detectable. The following bars show the measurements of the wells containing genomic DNA of the bacterium E. coli, the partial sequence of the original 13 contains. Here, the suppression of the hybridization and thus the suppression of the transmission change is greater, the more genomic DNA of the bacterium E. coli, the partial sequence of the original 13 Contains in the sample 12 is.

The features disclosed in the foregoing description, the claims and the drawings may be of importance both individually and in any combination for the realization of the invention in its various forms.

LIST OF REFERENCE NUMBERS

1

nucleic acid

2

reaction volume

3

first nanoparticles

4

oligonucleotide

5

primer sequence

6

spacer

7

Abasic modification

8th

primer

9

nanoparticles

10

Füllmolekül

11

DNA polymerase

12

sample

13

original

14

complement

15

forward primer

16

reverse primer

17

laser

18

light source

19

mirror scanner

20

mirror

21

first oligonucleotides

22

test probe

23

second nanoparticles

24

sample tubes

25

glass cuvette

26

water bath

27

diffuser

28

first laser

29

second laser

30

photodiode

31

test sequence

32

Multiwell plate

33

foil

34

first heating block

35

recess

36

second heating block

sequences

/iSp9/

= abasische Modifikation Spacer9

/ddC

= Dideoxycytidine

/ ISp9 /

= Abasic modification Spacer9

/ ddC

= Dideoxycytidines

This is followed by a sequence protocol according to WIPO St. 25. This can be downloaded from the official publication platform of the DPMA.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• US 4683202 [0002]
• WO 2007/143034 A1 [0003]
• US 2002/0061588 A1 [0004]
• US 2003/0143604 A1 [0005]
• DE 102012201475 [0006]
• EP 2162549 [0136, 0143]

Cited non-patent literature

• D. Renneberg and CJ Leumann, "Watson-Crick base-pairing properties of tricyclo-DNA", J. Am. Chem. Soc., 2002, Vol. 124, pp. 5993-6002 [0012]
• J. Hurst et al., Anal. Chem., 78 (24), 8313-8318, 2006 [0135]
• J. Hurst, supra [0136]
• J. Hurst, supra [0141]

Claims (24)

Use of one or more nanoparticles ( 9 ), each containing at least one oligonucleotide ( 4 ) are conjugated to duplicate nucleic acids ( 1 ), one or more of the oligonucleotides ( 4 ) at least one primer sequence ( 5 ) and a further section which extends from the nanoparticle end of the primer sequence in the direction of the nanoparticle, characterized in that the further section comprises at least one abasic modification ( 7 ) having. Use according to claim 1, characterized in that the abasic modification ( 7 ) at the end of the further segment facing the primer sequence adjacent to the primer sequence ( 5 ) is arranged. Use according to claim 1 or 2, characterized in that the abasic modification ( 7 ) 3'-side with respect to the primer sequence ( 5 ) is arranged. Use according to one of claims 1 to 3, characterized in that the abasic modification ( 7 ) is selected from the group comprising: 1 ', 2'-dideoxyribose, triethylene glycol and hexa-ethylene glycol. Use according to one of claims 1 to 4, characterized in that the further section comprises several abasic modifications ( 7 ) having. Method for duplicating nucleic acids ( 1 ) in a sample ( 12 ), the method comprising a amplification step for amplifying the nucleic acids and a test step for determining the concentration of the products of the amplification step and wherein the assay steps begin after the end of the amplification step, characterized in that substances are added to at least a portion of the sample in the assay step. Method for duplicating nucleic acids ( 1 ) in a sample ( 12 ), the method comprising a amplification step for amplifying the nucleic acids and a test step for determining the concentration of the products of the amplification step, characterized in that the sample ( 12 ) no substances are supplied in the test step. Method according to claim 6 or 7, characterized in that a global temperature of the sample ( 12 ) during the test step is different from a global temperature of the replication step. Method according to claim 6 or 7, characterized in that a global temperature of the sample ( 12 ) during the test step is substantially equal to a global temperature of the replication step. Method according to one of claims 6 to 9, characterized in that in the test step for determining the concentration of the products of the amplification step test probes ( 22 ) which contain a nanoparticle ( 23 ) exhibit. Process according to claim 10, characterized in that the nanoparticles ( 23 ) of the test probes ( 22 ) a size other than nanoparticles ( 9 in the amplification step for amplifying the nucleic acids ( 1 ) be used. Method for duplicating nucleic acids ( 1 ), characterized in that nanoparticles ( 8th ) in a reaction volume ( 2 ) transfer heat to their surroundings by excitation. A method according to claim 12, characterized in that an exposure time (t _A ) is shorter than 10 s. A method according to claim 12 or 13, characterized in that an exposure time (t _A ) is longer than 1 ps. Method according to one of claims 12 to 14, characterized in that a heating time of the nanoparticles ( 9 ) is shorter than 100 ms. Method according to one of claims 12 to 15, characterized in that a cooling time is shorter than 100 ms. Method according to one of claims 12 to 16, characterized in that a power density with which the nanoparticles ( 9 ) is more than 10 W / mm ² . Method according to one of claims 12 to 17, characterized in that a power density with which the nanoparticles ( 8th ), less than 20,000 kW / mm ² . Process according to any one of Claims 12 to 18, characterized in that the concentration of the amplicon to be multiplied in the process is greater than 10 ^-23 nM at the beginning of the process. Process according to any one of Claims 12 to 19, characterized in that the concentration of the amplicon to be multiplied in the process is less than 1 pM at the beginning of the process. Method according to one of claims 12 to 20, characterized in that the number of amplicons to be duplicated in the process at the beginning of the process is less than 500,000. A method for amplifying a nucleic acid by means of a polymerase chain reaction in which a cycle consisting of the steps of denaturing, annealing and elongation is repeated. A method according to claim 22, characterized in that the number of passes of the cycle of the polymerase chain reaction is greater than 45. A method according to claim 22 or 23, characterized in that at least one of the passes a cycle time t _{c is} shorter than 40 seconds.

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