Abstract
A reaction mixture for providing a reaction batch for performing a quantitative real-time PCR contains at least one target DNA, which at least in parts corresponds to the DNA section being quantified, at least one reference DNA of defined sequence and in a defined amount, at least two different fluorescent probes of different sequence which generate a signal at different wavelengths, primers, deoxynucleotides and a DNA polymerase. The target DNA and the reference DNA have the same primer binding sites and different probe binding sites. At least one of the fluorescent probes is intended for binding to a section of the target DNA outside the primer binding sites in the amplicon, and at least one of the fluorescent probes is intended for binding to a section of the reference DNA outside the primer binding sites in the amplicon.
Claims
1. A reaction mixture for providing a reaction preparation for performing a quantitative real-time polymerase chain reaction (PCR) for quantifying at least one deoxyribonucleic acid (DNA) segment, the reaction mixture comprising: at least one target DNA which corresponds at least in parts to the at least one DNA segment to be quantified; at least one reference DNA having a defined sequence and in a defined amount; at least two different fluorescent probes of different sequence that generate signals at different wavelengths; and primers, deoxynucleotides, and DNA polymerase, wherein the at least one target DNA and the at least one reference DNA have the same primer binding sites and different probe binding sites, wherein at least one of the fluorescent probes is intended for binding with a segment of the at least one target DNA outside the primer binding sites, and wherein at least one of the fluorescent probes is intended for binding with a segment of the at least one reference DNA outside the primer binding sites.
2. The reaction mixture as claimed in claim 1, wherein a GC content of the at least one target DNA and a GC content of the at least one reference DNA are identical with a deviation of up to 15%.
3. The reaction mixture as claimed in claim 1, wherein a base pair length of the at least one target DNA and of the at least one reference DNA are identical with a deviation of up to 15%.
4. The reaction mixture as claimed in claim 1, wherein the at least one target DNA, the at least one reference DNA, the primers, the deoxynucleotides, and/or the DNA polymerase are provided in lyophilized form.
5. The reaction mixture as claimed in claim 1, wherein the amount of the at least one reference DNA is present in a concentration which corresponds to a detection limit for the at least one DNA segment to be quantified.
6. The reaction mixture as claimed in claim 1, wherein: the mixture is used to detect and optionally to quantify the at least one DNA segment from a genome, and the reaction preparation contains the at least one target DNA and the at least one reference DNA in defined amounts in a ratio of 1:1.
7. A method for performing a quantitative real-time polymerase chain reaction (PCR), comprising: performing a PCR process using at least one reaction mixture, wherein a sample containing at least one deoxyribonucleic acid (DNA) segment to be quantified is added to the at least one reaction mixture; and capturing fluorescent signals of at least two fluorescent probes, wherein the at least one reaction mixture comprises: at least one target DNA which corresponds at least in parts to the at least one DNA segment to be quantified; at least one reference DNA having a defined sequence and in a defined amount; the at least two different fluorescent probes which are of different sequence and generate the fluorescent signals at different wavelengths; and primers, deoxynucleotides, and DNA polymerase, wherein the at least one target DNA and the at least one reference DNA have the same primer binding sites and different probe binding sites, and wherein at least one of the fluorescent probes is intended for binding with a segment of the at least one target DNA outside the primer binding sites, and wherein at least one of the fluorescent probes is intended for binding with a segment of the at least one reference DNA outside the primer binding sites.
8. The method as claimed in claim 7, further comprising: ascertaining an assay result from a ratio of the fluorescent signals of the fluorescent probes.
9. The method as claimed in claim 7, further comprising: performing the method in a PCR array comprising a plurality of array vessels.
10. The method as claimed in claim 7, further comprising: using the method for a nested PCR process comprising a preamplification and at least one downstream detection reaction; and estimating an amount of PCR product of the preamplification by the quantification.
11. The method as claimed in claim 10, wherein: a first primer pair is used for the preamplification and at least one second primer pair is used for the at least one downstream detection reaction, the at least one target DNA and the at least one reference DNA each have complementary sequence segments in relation to the primer sequences, and the complementary sequence segments in relation to the at least one second primer pair lie within the segment between the complementary sequence segments in relation to the first primer pair.
12. The method as claimed in claim 10, further comprising: using the nested PCR process for a point mutation detection; and using a mutation-sensitive primer and/or a mutation-sensitive fluorescent probe for the at least one downstream detection reaction.
13. The method as claimed in claim 10, wherein: the nested PCR process is a multiplex process for detecting at least two particular gene segments in a genome, what is performed for a quantification of the preamplification is a control reaction in which a control exon from the genome is amplified, the at least one target DNA and the at least one reference DNA are configured to match with the control exon, and what are deduced from the quantification of the amplification of the control exon are the amounts in a case of the amplification of the gene segments to be detected during the preamplification.
14. A kit for performing a quantitative real-time polymerase chain reaction (PCR) for quantifying at least one deoxyribonucleic acid (DNA) segment, the kit comprising: at least one target DNA which corresponds at least in parts to the at least one DNA segment to be quantified; at least one reference DNA having a defined sequence and in a defined amount; at least two different fluorescent probes of different sequence that generate signals at different wavelengths; and optionally primers, deoxynucleotides, DNA polymerase, and/or buffer components, and wherein the at least one target DNA and the at least one reference DNA have the same primer binding sites and different probe binding sites, wherein at least one of the fluorescent probes is intended for binding with a segment of the at least one target DNA outside the primer binding sites, and wherein at least one of the fluorescent probes is intended for binding with a segment of the at least one reference DNA outside the primer binding sites.
15. The kit as claimed in claim 14, wherein a GC content of the at least one target DNA and a GC content of the at least one reference DNA are identical with a deviation of up to 15%.
Description
[0023] In the figures:
[0024] FIG. 1 shows a schematic representation of the design of the target DNA and the reference DNA to illustrate the basic principle of the concept for performing a quantitative real-time PCR;
[0025] FIG. 2 shows a schematic representation of the template DNAs used for the quantitative real-time PCR and a schematic representation of possible experimental results in the quantification of a particular DNA segment in a sample;
[0026] FIG. 3 shows a schematic representation of the DNA templates used for a quantitative real-time PCR (FIG. 3A) and a schematic representation of possible experimental results (FIG. 3B) in the application of the concept in the context of a quantitative nested PCR;
[0027] FIG. 4 shows a schematic representation of possible designs for a reference DNA in the context of a point mutation assay;
[0028] FIG. 5 shows a schematic representation of the template DNAs used to elucidate a multiplex embodiment of a nested PCR and
[0029] FIG. 6 shows a schematic representation of the implementation of the quantitative real-time PCR in a microfluidic PCR array.
DESCRIPTION OF EXEMPLARY EMBODIMENTS
[0030] FIG. 1 elucidates the basic principle of the design of the template DNAs used, i.e., the target DNA 11 and the reference DNA 12. What can be used as the basis for the PCR reaction preparation is a classic TaqMan® system, with use of two different fluorescent probes, as elucidated at the start. Here, the target DNA corresponds to the DNA sequence to be actually analyzed or quantified, for example the DNA sequence of a gene segment. The target DNA 11 is supplemented with an artificial reference DNA which has a defined sequence and which is used in a defined amount. The target DNA 11 and the reference DNA 12 have the same primer binding sites, i.e., respectively a binding site 13 for the forward primer and respectively a binding site 14 for the reverse primer. In the rest of the base pair sequence 15, 16, these template DNAs 11, 12 differ. In particular, they have different binding sites 17, 18 for the probes used. The two template DNAs 11 and 12 are also referred to as quanticon 11 for the amplicon to be quantified and as articon 12 for the artificial amplicon. The following table summarizes the design of the quanticon (target DNA) 11 and the articon (reference DNA) 12:
TABLE-US-00001 Quanticon Articon Forward primer Target-specific Target-specific Probe Target sequence, Orthogonal sequence, fluorophore of fluorophore of color A color B Sequence Target sequence Orthogonal sequence Reverse primer Target-specific Target-specific
[0031] The fluorophores of the fluorescent probes are chosen such that the two colors are distinguishable from one another by means of a detector (filter set). The orthogonal sequence 16 of the reference DNA 12 is expediently nonidentical in relation to the target sequence 15 of the target DNA 11. The GC content should be as identical as possible to the GC content of the target sequence 15 of the target DNA. The base pair length of quanticon, i.e., target DNA 11, and articon, i.e., reference DNA 12, should be of the same length as well. As a result, the melting temperatures of the two amplicons 11, 12 are very similar, and so the same amounts of amplified material arise in principle in an efficient PCR. Using these template DNAs, a quantitative real-time PCR is carried out, with amplification of quanticon 11 and articon 12 in the same reaction vessel as an effectively duplex reaction. Meanwhile, the two probes are recorded, for example after each PCR cycle or continuously. Here, the articon 12 can be initially charged in a predefined amount in the region of or above the detection limit and must be detected as signals of the probe B in the event of a successful PCR. The articon serves in this case as a reaction control. The amplification of the quanticon 11 and of the gene segment (target) to be quantified possibly additionally present in the reaction preparation is detectable as a signal of probe A. The signals of probe A and B are, then, in defined ratios. If the same starting amount of articon 12 and quanticon 11 is present, the two amplification curves are congruent. If more quanticon 11 is present, it is detected earlier and the curve of the articon 12 follows depending on the concentration thereof. This can be calculated by means of the reaction efficiency and from the firmly defined amount of the articon 12. The initially charged amount of articon 12 is, then, an absolute reference point, the amount of which is known. The efficiency of the reaction can be ascertained by means of the curve shape of the exponential phases. As a result, the unknown starting amount of the quanticon 11 can be calculated using the absolute reference point.
[0032] FIG. 2 shows the implementation of the reaction system for the case of the sample material (sample) being present as a genome. For example, this may be used if particular gene segments from a lysate are to be detected. Comparable with the principle from FIG. 1, what is used here is a quanticon 11 (Q) and an articon 12 (A). Additionally present in the reaction preparation is the gene segment 20 (S) to be amplified, as cell lysate containing genetic material which is formed by the genome of the cell(s). In this case, the quanticon 11 and the articon 12 are initially charged in a predefined amount in a ratio of 1:1. The amount chosen can, for example, lie in the proximity of above the detection limit. Another possibility is to adjust the amount to an ideally functional range for the PCR reaction, so that the PCR proceeds particularly efficiently. In general, every quantitative real-time PCR (qPCR) has limits within which the reaction proceeds efficiently. In this connection, the detected C.sub.T values, which describe the start of the exponential growth of a curve, are in a linear ratio to the logarithmized starting amount used. If the amount used of the quanticon 11 and the articon 12 is chosen in this range, a signal should be detected with each successful PCR process. The signal of the articon 12 is the signal which must be measured last in chronological order. If it is missing, the reaction control is negative. If the signal of the quanticon 11 is detected simultaneously with the signal for the articon 12, this means that only quanticon 11 and articon 12 was present in the reaction mixture and no sample 20. This case is represented in graph A of FIG. 2 and serves as a detection control for the function in principle of the PCR preparation. Here, the lines 11 and 12 represent the respective fluorescent signals of the quanticon 11 (fluorophore A) and the articon 12 (fluorophore B). If the same DNA segment as in the quanticon 11 was present in the genome or in the sample 20, said segment from the sample 20 is concomitantly highly amplified. The result of this is that the signal of the quanticon 11 (graph B in FIG. 2) is detected earlier, the detected signal being composed of the amplification of the quanticon 11 and the sample 20. As already elucidated in the principle from FIG. 1, it is possible, then, to calculate the starting amount of the DNA segment to be tested from the sample 20 (sample). The predefined amounts of articon 12 and quanticon 11 provide, then, not only the absolute reference point to calculate the quantification, but also ensure signals and serve as control.
[0033] In a further embodiment of the PCR process, the process can be performed dynamically, by the signal of the quanticon 11 representing a termination criterion for the reaction, so that the PCR process can be ended after the appearance of the signal of the quanticon 11. Since the amount of the quanticon 11 can be transferred into an efficient range for the PCR process, what is possible is a detection approximately in the temporal middle of the planned process duration, i.e., in middle cycle numbers. In the event of a positive sample, i.e., the sought DNA segment in the sample 20 is present, the signal of the quanticon 11 (together with the signal of the sample 20) lies before the signal of the articon 12, and so the process time for measurement can be shortened. In this case, only a qualitative statement is possible after the termination of the reaction.
[0034] FIG. 3A and FIG. 3B illustrate the described concept in the context of a qualitative nested PCR. Such a PCR method can, for example, be used for detection of mutations. To this end, what is highly copied from the genomic DNA 30 of cells is the target region in which the mutation lies. Thereafter, the ratio of wild type to mutation is measured. For this purpose, a preamplification is upstream of the actual detection reaction in order to ensure that sufficient material is present for a detection. This is especially of great significance if little cell material is present, such as, for example, in the case of a liquid biopsy containing circulating tumor cells. As illustrated in FIG. 3A, the presently described concept is implemented in such a system such that the locus of the mutation 35 (target DNA) is initially highly copied in a sufficiently large segment using a defined primer pair. For this first primer pair, what are present on the genomic DNA of the sample 30 are corresponding binding sites 33, 34 for a forward primer and a reverse primer. For the control, monitoring and quantification of the process, what is chosen is a probe binding site 37 for a first fluorescent probe A in immediate proximity of the primer binding site 33. Designed congruently with this amplicon in the sample 30 is a quanticon 21, i.e., a target DNA 21 which has corresponding primer binding sites 23 and 24 and a corresponding probe binding site 27 for the fluorescent probe A. Additionally designed is an articon 22 (reference DNA) having the same primer binding sites 23, and a differing probe binding site 28 for a fluorescent probe B. These components 30, 21, 22 provide the basis of the preamplification, which is quantifiable according to the principle elucidated by means of FIG. 2. Furthermore, for the subsequent detection reaction 102, what are provided are further primer binding sites 43, 44 for a further primer pair comprising a second forward primer and a second reverse primer, the binding site 43 for the second forward primer being joined to the binding site 37 for the fluorescent probe A in the case of the genomic gene segment 30. The binding site 44 for the second reverse primer is situated downstream of the actual target DNA 35 which represents the gene segment to be detected. Situated on the reference DNA 22 (articon for the preamplification) are corresponding primer binding sites 43, 44. Provided on the target DNA 21 (quanticon for the preamplification) are differing, i.e., orthogonal, sequences at the positions 143, 144 which correspond to the primer binding sites 43, 44 of the articon sequence 22. The sequence between the sequences 143, 144 on the quanticon sequence 21 corresponds to the target DNA sequence 35 of the DNA segment to be quantified of the sample 30.
[0035] After the preamplification 100, which is carried out after addition of the first primer pair, what is present as PCR product is the amplified gene segment 30′ to be quantified (amplified sample). Additionally present is the amplified articon 22′. The likewise amplified quanticon 21 substantially corresponds, from the sequence, to the amplified sample 30′. Within the primer binding sites 43, 44 for the second primer pair of the subsequent detection reaction 102, what is situated after the primer binding site 43 for the forward primer is a binding site for a further fluorescent probe A′ which is used in the subsequent detection reaction 102. Correspondingly, the articon 22 or the amplified articon 22′ has, after the primer binding site 43, a different probe binding site 48 for a further fluorescent probe B′, likewise for the subsequent detection reaction 102. On the articon 22 or the amplified articon 22′, what follows is an orthogonal sequence 26 which is orthogonal in relation to the target sequence 35 of the sample 30 to be tested. What follows is the binding site 44 for the reverse primer of the subsequent detection reaction 102 and the binding site 24 for the reverse primer from the preamplification. The GC content and the length of the base pair sequences among articon 22 and the corresponding segment in the sample 30 and the quanticon should approximately correspond, as already explained above.
[0036] The quantification of the preamplification 100 is, in principle, carried out as already elucidated by means of FIG. 2 and is illustrated in the top part of FIG. 3B. The signals of the probes A and B are both depicted here. Graph A shows the case of overlapping of the signal of the quanticon 21 (probe A) and the signal of the articon 22 (probe B). In this case, there is no DNA segment to be detected in the sample 30. Graph B shows the case of the signal of the amplified quanticon 21 together with the amplified gene segment from the sample 30 appearing chronologically before the signal of the amplified articon 22. In this case, the sought gene segment in the sample 30 is present. If no sample can be detected (graph A), the entire run can be stopped and what can be output is that no detection occurred (negative assay result). If, as per graph B, sample is detected, the PCR process can be continued until the articon 22 is detected. Then, the process can optionally be terminated. Alternatively, a predefined number of PCR cycles can also be executed. From the amplification curve of the sample 30 together with the quanticon 21, it is possible to calculate efficiency. By means of the predefined articon 22, it is possible to calculate the end concentration and the start concentration of all amplified materials. On this basis, the preparation can be diluted and be prepared with a new master mix (step 101) in such a way that the preparation corresponds to the ideal starting concentrations for the subsequent detection assay(s) (step 102). The dilution can be done by hand, for example when the reactions take place in a bulk system, for example a classic qPCR cycler. Preferably, the process is carried out in a fully automated liquid handler, microfluidic systems being particularly suitable. Here, liquids can be diluted and distributed with the aid of microfluidic pumping and aliquoting systems.
[0037] For the actual detection reaction 102, for example for a point mutation detection, the reaction preparation is amplified using the primers required for this purpose (second primer pair) and the signal course of the probe A′ (curve 470) and the probe B′ (curve 480) is observed and evaluated. According to the reaction concept, the articon 22′ is outnumbered, i.e., less starting material of the articon 22′ is present than starting material of the sample 30′. This is because more sample amplicon, consisting of the amplified sample 30′ and the amplified quanticon 21, arises in the preamplification 100. Therefore, a dynamic termination of the PCR after immediate detection is highly advantageous, since the articon 22′ and the sample 30′ in the exponential phase make the estimation of the amplicon amounts more accurate than in the case of a detection in the saturation phase. Since, then, the articon 22′ is again present in defined amounts and the sample 30′ acts as a new quanticon, the second qPCR, i.e., the detection reaction 102, can also be completely quantified. The number of copies from the start up to the end of the process is thus known. The amplified quanticon 21 of the first reaction (preamplification 100) does not come into consideration in the second reaction (detection reaction 102), since the corresponding positions 143, 144 in relation to the primer binding sites 43, 44 on the target DNA sequence 21 of the preamplification were chosen orthogonally, i.e., differingly.
[0038] For the detection reaction 102, it is possible to add to the master mix thereof additionally a further articon. Here, instead of the articon from the first preamplification, what is added is a new articon for the second detection reaction. This is useful, since the first reaction mixture is generally diluted and therefore the articon (but not the increased actual sample) is detected. Therefore, after the dilution, a defined amount of articon is added again for a more accurate determination. This further articon can, for example, be prestored in a (second) lyobead required for the detection reaction. This is especially advantageous for determining the ratio of wild type and mutation type in a point mutation detection. A further quanticon which has the same primer binding sequences is also used. The amplified material of the first reaction 100 must then be diluted such that said reaction corresponds to the concentration of the initially charged, second quanticon.
[0039] FIG. 4 shows embodiments for a possible design of the articons (reference DNA) 52, 62 for a point mutation detection. Said articons 52, 62 are intended for the application of a nested PCR in the context of a point mutation assay. Generally, two general PCR detection strategies are used in point mutation detections. What are chosen here are either mutation-sensitive primers (a) or mutation-sensitive probes or blockers (b). In method (a), the primer is designed such that it can only bind when the mutation is present. Examples thereof are so-called ARMS (amplification refractory mutation system) systems. In method (b), what is used is a mutation-sensitive probe or blocker which only binds when the mutation is present (e.g., PNA-CLAMP systems—peptide nucleic acid (PNA)-mediated PCR clamping; H. Ørum et al., Nucleic Acids Res. 21: 5332-5336, 1993). However, since these bindings are not 100% efficient, a reference signal in relation to the wild type is concomitantly measured. Therefore, for the implementation of the concept according to the invention for such a detection, it is useful to involve a second quanticon. For the implementation, the mutation 301 is incorporated into the orthogonal sequences for the articon 52, 62. The binding site of the mutation should therefore be included. In version 62 with a mutation-sensitive primer, the articon therefore comprises the following segments: binding site 23 for the first forward primer, binding site 28 for the probe B, binding site 63 for a mutation-specific primer which represents the forward primer of the second primer pair for the detection reaction 102, an orthogonal sequence 26, a binding site 44 for the reverse primer of the detection reaction 102 and a binding site 24 for the reverse primer of the preamplification. For a detection system with a mutation-sensitive blocker or a mutation-sensitive probe, the articon 52 is designed such that the binding site for this probe or the blocker comprises the mutation site 301 at exactly the same site as in the mutation type. Apart from that, the articon 52 corresponds to the articon 62 or the articon 22. Using such a construct, it is possible, then, to measure in the reaction a signal for a reaction with 100% wild type (second quanticon) and a signal for 100% mutation (articon 52 or 62) and to compare them with the sample. This is all possible within one reaction preparation, thereby allowing an extreme simplification of full automation, for example in a point-of-care application. It is particular advantageous here when the corresponding master mixes are initially charged as lyophilisates.
[0040] FIG. 5 shows a multiplex embodiment of a nested PCR, wherein two detection reactions can be performed in one preparation. In said embodiment, the starting point is a lysate composed of few cells, for example 10 to 1000 cells. What can be present in said lysate are, for example, cells from an enrichment, for example from an enrichment of circulating tumor cells or from an enrichment of immune cells, for example specific T cells, from a body fluid, such as blood, urine, spinal fluid or other. Said cells are lysed in a small volume and provide the sample for the performance of the method. In said cell lysate, two gene segments 70, 80 are to be detected, i.e., for example an exon A (gene segment 70) and an exon B (gene segment 80). The gene segments 70, 80 are initially amplified (preamplification), so that it is possible in the downstream step to detect, in one or more detection reactions, anomalies on said gene segments such as mutations or function-typical gene sequences, for example the genetic coding of an antigen epitope. In the first step of the preamplification, the two target exons, i.e., the gene segments 70 and 80, are first amplified from the genetic material of the lysed cells. In addition, a sample control is run. For the sample control, a control exon C is amplified as gene segment 90. Here, this can, for example, be an exon of the sought gene, on which the anomaly is not present. If it is amplified, the reaction is considered successful and to be evidence of the presence of the sample material, i.e., genetic material, in the sample. For the detection and the quantification of the amplification of the control exon 90, what are correspondingly used as already described are a target DNA (quanticon) 21 and a reference DNA (articon) 22 which are, from their structure and their components, matched with the control exon 90 according to the above-described principles. If the amplicons 70, 80 and 90 all have approximately the same length and the same GC content, then what should arise in the reaction preparation in a triplex reaction are about the same number of copies for each template. If there are deviations in the length and in the GC content, conserved ratios of the amplified amplicons ensue, it being possible for the ratios to be additionally influenced by the DNA structure and epigenetic modifications. This means that, even if the reaction is possibly less efficient in the case of the amplification of one of the exons, the ratios of the amplicon copies which arise are nevertheless constant. This allows measurement of just one amplicon, namely the control exon C, with respect to a quantification, and from this, it is possible to determine the amplicon number for all exons. A complicated three-probe design is thus not necessary; instead, it is generally sufficient to use just one two-probe system for the quantification of the control exon C (gene segment 90). Therefore, the preamplification is initially quantified by the control exon 90, and so the amplified materials can be estimated for the subsequent detection reaction as described above and optionally be distributed and diluted in situ for optimal reaction conditions in the detection reaction. After distribution and dilution, a new master mix is added which is intended for the specific assay of the detection reaction and which can likewise be quantified as per the remarks in relation to FIG. 2.
[0041] The design for the individual amplicons preferably looks as follows: Exon A (gene segment 70) has, on the periphery of the amplicon, the binding sites 71, 72 for the primers of the preamplification. Exon B (gene segment 80) and the control exon C (gene segment 90) have corresponding primer binding sites 81, 82 and 91, 92, respectively, but with different sequences. In the case of the exons A and B to be tested in the subsequent detection reaction, what follows in each case is the binding site for the primers of the second reaction (detection reaction) 73, 74 and 83, 84, respectively, on the gene segments 70 and 80, respectively. If a probe is intended for the detection reaction, the binding site thereof is included in this sequence. The control exon C (gene segment 90) has, after the primer binding site 91, the binding site 97 for a fluorescent probe A. Correspondingly as elucidated by means of FIG. 2, the quantification of the control exon C (gene segment 90) is achieved by supplementing a quanticon or target DNA 21 and an articon or reference DNA 22 that are provided with the same primer binding sites 91, 92 as the control exon C (gene segment 90). The target DNA 21 has furthermore the same binding site 97 for the probe A. The reference DNA 22 likewise has a probe binding site 98, but with a different sequence for binding a fluorescent probe B. From the signals to be generated with this reaction preparation, the resultant copies N.sub.C and the starting quantity N.sub.O e deduced. From the conserved ratios, the amounts of the exons A and B (gene segments 70 and 80) are calculated. Said ratios can be worked out as part of assay development. The ratios are specific for the assay in question. The ratios are intrinsically constant, but must be measured, i.e., parameterized, for each application. According to the method elucidated by means of FIG. 2, the master mixes of the subsequent two separate and parallelly processible specific detections for the exon A and for the exon B can each have a quanticon and an articon that have the same primer binding sites as the respective target exon A and B. For the detection of point mutations, it is possible, as elucidated by means of FIG. 4, for the quanticon to have the wild-type sequence and the articon to have the mutant sequence. The middle part of the representation in FIG. 5 schematically represents the entire reaction procedure. What takes place first is the preamplification 200, with the presence of exon A, exon B, control exon C and the quanticon and the articon as templates in the preparation. After the performance of the amplification reactions, what is obtained is the quantification result 210 (N.sub.0 for control exon C, N.sub.C for control exon C; calculable therefrom: N.sub.C, N.sub.0 for exon A, N.sub.C, N.sub.0 for exon B). To achieve optimal starting conditions for the subsequent detection reaction for the exon A and the exon B, the optimal concentrations of exon A (N.sub.S, 2 exon A) and exon B (N.sub.S, 2 exon B) are set in step 220 by distribution and optionally dilution of the preparations. Thereafter, by addition of the master mixes for the respective detection reactions, what is performed in step 230 is, for example, the respectively specific mutation detection, with not only the exon A and the exon B, but in each case a correspondingly designed quanticon A and articon A and quanticon B and articon B, respectively, being added for this purpose, as illustrated in the bottom part of FIG. 5.
[0042] FIG. 6 illustrates the implementation of the described PCR concept in a microfluidic qPCR array 500. The array 500 is, for example, integrated into a chip composed of structured silicon, said array 500 being situated in a microfluidic chamber which is provided with an inflow 501 and an outflow 502. In one possible embodiment, individual reaction vessels of the array 500 can be actuated, or admixed with liquids, in a global manner. For example, a preamplified sample can be flushed across the array 500, so that the individual reaction vessels of the array 500 are filled. By means of a seal, it is possible to prevent communication via diffusion between the individual reaction vessels in a second fluidic step. If, then, each reaction vessel of the array 500 is, for example, prespotted with a lyophilized master mix and/or with the primers and the probe sequences, what is possible by means of the method with an n×m array is a maximum n×m degree of multiplexing including a quantification and quality control. The reaction preparations according to the concept of the invention can preferably be developed on the basis of TaqMan® systems. The synthesis of the individual template DNAs, especially the quanticons and the articons, can be done using customary nucleic acid synthesis. Preferably, the master mixes including the template DNAs can be prestored as lyophilisates. Microoptofluidic systems in particular are suitable for an automation of the processes.
