Method and apparatus for pre-amplification of a nucleic acid mixture

Abstract

A method for pre-amplification of a nucleic acid mixture (30) is described, comprising the following steps - Denaturing (21) the nucleic acid mixture (30) to obtain single-stranded nucleic acids (31, 32), - Hybridization (22) of the single-stranded nucleic acids (31, 32) with chemically modified primers with random sequence (41) or with chemically modified primers with random sequence (41) and with chemically modified primers with specific sequence (42), wherein the chemically modified primers with random sequence (41) or the chemically modified primers with random sequence (41) and the chemically modified primers with specific sequence (42) have a Self-Avoiding Molecular Recognition Systems (SAMRS) modification, and - isothermal pre-amplification (23) of the hybridized nucleic acids (51-54) in particular as multiple displacement amplification (MDA) by means of a strand displacement (SD) polymerase (61).

Description

[0001] The present invention relates to a method for pre-amplifying a nucleic acid mixture. The present invention also relates to a microfluidic device configured to perform pre-amplification using the method. Furthermore, the present invention relates to the use of chemically modified primers with random sequence (41) featuring a Self-Avoiding Molecular Recognition System (SAMRS) modification. State of the art

[0002] In molecular diagnostics, diseases can be detected based on nucleic acids that function as biomarkers. These nucleic acids can be DNA or RNA. To detect as many pathogens as possible simultaneously using such biomarkers, probe-based methods or sequencing can be employed. However, these methods require pre-amplification of, for example, the relevant biomarkers or the entire sample mixture when the sample size is small. Pre-amplification serves to multiply the nucleic acids before more detailed analysis is performed. This is particularly useful when the initial amounts of the (target) nucleic acids are very low and insufficient to obtain reliable results. Further amplification steps may follow.

[0003] For reasons of cost, time, and efficiency, it is desirable to analyze many genetic DNA or RNA segments simultaneously, for example, in multiplex PCR. This involves using many sets of primers in a single reaction. As a result, in addition to desired binding products, undesired interactions between these primers themselves can occur, leading to the formation of unwanted, target-independent byproducts.

[0004] The use of Self-Avoiding Molecular Recognition Systems (SAMRS)-modified primers is already known, but only for the detection of certain target nucleic acid sequences.

[0005] In DE102022211087A1 a method for the isothermal pre-amplification of a nucleic acid mixture using an SD polymerase is described. Disclosure of the invention

[0006] Methods for the universal pre-amplification of nucleic acid mixtures are highly complex systems and difficult to control, which is why changes, for example by adding further components or modifying primers, are not easily possible and must be carefully considered in order to prevent unwanted or unpredictable side reactions as well as a failure of the target reaction.

[0007] Another challenge is that high initial concentrations of nucleic acids quickly lead to saturation of the reaction, which may prevent a sufficient amplification factor of the target nucleic acids from being achieved.

[0008] The inventive method for pre-amplifying a nucleic acid mixture, which is contained in particular in a patient sample, comprises denaturing the nucleic acid mixture to obtain single strands of the nucleic acids. The single strands of the nucleic acids are then hybridized with chemically modified primers having a random sequence.

[0009] In this case, the chemically modified primers with random sequence exhibit a Self-Avoiding Molecular Recognition Systems (SAMRS) modification.

[0010] The hybridized nucleic acids thus obtained are subjected to isothermal pre-amplification, in particular as multiple displacement amplification (MDA), using an SD (strand displacement) polymerase.

[0011] A key advantage of using SAMRS-modified primers with random sequences is that the SAMRS modification prevents unwanted bonding between the primers themselves. When two such modified primers meet, fewer hydrogen bonds form between their nucleobases compared to unmodified primers. However, the binding of SAMRS-modified primers to the DNA and / or RNA to be amplified is not affected by the modification.

[0012] This is particularly advantageous for applications requiring general, universal pre-amplification of nucleic acids, for example, from different species, such as in whole genome amplification (WGA) and / or whole transcriptome amplification (WTA), because the SAMRS modification achieves maximum suppression of side reactions and unwanted target-independent binding products. The primers cannot interact or bind to each other and are therefore available for the reaction to be carried out.

[0013] The SAMRS modification is introduced, for example, into a certain number of nucleobases of the chemically modified primers, for example into three nucleobases per primer, advantageously near their 3' ends.

[0014] In this process, for example, an andenine is replaced by 2-aminopurine-2'-deoxyriboside and / or a thymine by 2'-deoxy-2-thiothymidine and / or a guanine by 2'-deoxyinosine and / or a cytosine by N4-ethyl-2'-deoxycytidine.

[0015] In a particularly preferred embodiment of the method, primers with a specific sequence are used in addition to the modified primers with random sequences to hybridize the single-stranded nucleic acids. The additional use of the specific primers enables semi-specific multiple displacement amplification (ssMDA). To prevent the chemically modified primers with random sequences from interacting with, for example, the specific primers also used, or the specific primers from interacting with each other, the chemically modified primers with specific sequences are also, for example, SAMRS-modified. This is particularly advantageous because universal pre-amplifications often involve a high nucleic acid background, such as a high human nucleic acid background when detecting nucleic acid targets from bacteria, viruses, fungi, and / or parasites.

[0016] When using SAMRS-modified primers at high initial concentrations of nucleic acids in the nucleic acid mixture to be pre-amplified, a significantly higher amplification factor of the target nucleic acids is achieved because the nucleic acid equilibrium is shifted towards the desired sequences during pre-amplification. Thus, high amplification factors of the target nucleic acids are achieved even against a high nucleic acid background, as the primers used do not interact with each other unintentionally and are thereby consumed, but are available for the intended reaction. Target amplification therefore proceeds preferentially and accelerated.

[0017] SD polymerase is a DNA polymerase suitable for isothermal amplification reactions and is already known for high strand displacement activity in amplification reactions.

[0018] The method is based on the finding that SD polymerases are also suitable for pre-amplification reactions. The use of SD polymerase offers several advantages. It enables isothermal pre-amplification without exhibiting the low processivity of mesophilic enzymes. Furthermore, some SD polymerases tolerate high temperatures during isothermal pre-amplification, which increases the reaction rate to such an extent that a significant reduction in reaction time is possible. Finally, some SD polymerases can use RNA as a template for amplification in addition to DNA. The method therefore requires little to no adaptation to different pathogens or biomarkers being detected, as it can universally amplify DNA and RNA, regardless of the application.

[0019] Prior to denaturation, the nucleic acid mixture can be detached from a solid phase, such as silicon dioxide, using water or an elution buffer. Denaturation is preferably carried out by heating the nucleic acid mixture dissolved in water, particularly preferably to a temperature in the range of 75°C to 98°C and most preferably to a temperature in the range of 80°C to 95°C. Heating is preferably carried out for a period of more than 10 seconds, and particularly preferably for a period in the range of 30 seconds to 120 seconds. This reliably dissolves the secondary and tertiary structures of the nucleic acids.

[0020] The chemically modified primers with random sequences and SAMRS modification, and in particular the chemically modified primers with specific sequences and SAMRS modification, for hybridizing the single-stranded nucleic acids, can preferably be combined with the nucleic acid mixture before denaturation, especially preferably by being dissolved in the water or elution buffer. They are insensitive to the denaturation conditions. The hybridization then preferably takes place at a temperature in the range of 4°C to 50°C for a period of time that is preferably more than 10 seconds. Under these conditions, these primers can bind to the single-stranded nucleic acids. The temperature required for hybridization is achieved, in particular, by cooling a solution of the single-stranded nucleic acids obtained by denaturation.

[0021] To prepare for isothermal pre-amplification, the solution of hybridized nucleic acids is treated with SD polymerase. Furthermore, deoxynucleoside triphosphates (dNTPs) and buffer components can be added. Alternatively, the dNTPs and buffer components can also be added before denaturation. The deoxynucleoside triphosphates serve as nucleic acid precursors in the pre-amplification. The buffer components serve to adjust the pH to an optimal value for pre-amplification. It is also preferred to add single-stranded binding proteins (SSBs). These proteins bind to single-stranded DNA to facilitate isothermal pre-amplification by supporting the SD activity of the polymerase.Furthermore, helicases, which are known to separate double-stranded nucleic acids into single-stranded nucleic acids, can be added. These proteins can further support the SD activity of the polymerase.

[0022] Isothermal pre-amplification is preferably started only after a homogeneous reaction mixture has formed from the solution of hybridized nucleic acids and all other added reagents. Isothermal pre-amplification is preferably carried out at a constant temperature in the range of 40°C to 72°C. A preferred pre-amplification period is in the range of 10 to 120 minutes. The duration of pre-amplification depends on the temperature during pre-amplification, as well as the temperature optimum and the rate of the polymerase used. The higher the temperature optimum of the polymerase and the higher the temperature during pre-amplification, the faster the process proceeds and the shorter the pre-amplification period can typically be. Compared to pre-amplification with mesophilic enzymes at 40°C or below, this can result in a significant reduction in reaction time.The reaction is significantly accelerated, particularly at temperatures between 45°C and 65°C. This method also has the advantage of being suitable for RNA pre-amplification even at such high temperatures, whereas other methods only allow RNA pre-amplification at lower temperatures.

[0023] In an advantageous embodiment, the chemically modified primers with random sequence and / or the chemically modified primers with specific sequence according to the invention further comprise a modification selected from the group consisting of LNA (Locked Nucleic Acid), MGB (Minor Groove Binder), C-5-propynyl deoxycytidine, C-5-propynyl deoxyuridine, aminoethyl phenoxazine deoxycytidine, 5-methyl deoxycytidine, 2-amino deoxyadenosine, trimethoxystilbene, pyrene, and spermine. A ZNA (Zip Nucleic Acids) modification is particularly preferred. These are spermine-modified primers. They have the advantage that they form particularly temperature-stable hybridized nucleic acids with single-stranded nucleic acids and that higher hybridization temperatures can be used without requiring sequence elongation of the primers. Furthermore, they reduce the self-hybridization of the primers when the primers are adequately loaded with spermine.This is particularly advantageous when the primers used are short and the isothermal pre-amplification is carried out at temperatures of at least 45°C.

[0024] Chemically modified primers with random sequences are also called random primers. They have a defined chain length, preferably between 6 and 15 bases, and consist of a purely random sequence. Their advantage lies in their high probability of covering all regions of the nucleic acid template to be amplified. They enable isothermal pre-amplification as uMDA (universal multiple displacement amplification).

[0025] In a particularly preferred embodiment of the method, primers with a specific sequence are used in addition to the modified primers with random sequences to hybridize the single-stranded nucleic acids. The additional use of the specific primers enables semi-specific multiple displacement amplification (ssMDA). These primers with specific sequences can carry, in addition to an optional SAMRS modification, other modifications. In particular, these are spermine groups. Primers with specific sequences and a ZNA modification are especially preferred; these primers, when appropriately loaded, exhibit mutual repulsion. This modification further facilitates the prevention of unwanted interactions between the primers with specific sequences.Furthermore, the primer length can be shortened while maintaining the desired melting temperature, making base pairing between the modified primers even less likely. This also allows for the use of a large number of different primers with specific sequences.

[0026] Specific-sequence primers are defined as primers that possess not only a defined chain length but also a defined sequence. This sequence can be specifically chosen to bind to particular loci on the nucleic acids, for example, flanking one or more targets, thereby promoting the enrichment of one or more targets relative to the background. Alternatively or additionally, the sequence can be chosen to bind to nucleic acid sequences for which SD polymerase exhibits lower processivity compared to other nucleic acid sequences, for example, due to challenging sequences with high GC content. By enhancing the pre-amplification of these otherwise insufficiently amplified sequences, not only is a more universal but also a more uniform pre-amplification is achieved.

[0027] In a particularly preferred embodiment, the chemically modified primers with random sequence have a SAMRS modification and the chemically modified primers with specific sequence also have a SAMRS modification.

[0028] In a further particularly preferred embodiment, the chemically modified primers with random sequence have a SAMRS modification and the chemically modified primers with specific sequence have a SAMRS modification as well as a ZNA modification.

[0029] In another particularly preferred embodiment, the chemically modified primers with random sequence have a SAMRS modification and a ZNA modification, and the chemically modified primers with specific sequence have a SAMRS modification.

[0030] In another particularly preferred embodiment, the chemically modified primers with random sequence have a SAMRS modification and a ZNA modification, and the chemically modified primers with specific sequence also have a SAMRS modification and a ZNA modification.

[0031] The primers with a specific sequence preferably contain at least one selection marker, such as biotin. This selection marker is advantageous when the primers are specific for a target and not merely used to increase uniformity. In this case, it is possible to purify or enrich the amplification products of the specific primers using their selection markers.

[0032] It is advantageous for both randomly sequenced and specific-sequence modified primers to have a single-strand break site. After the single-strand break is created by a suitably added endonuclease (nicking enzyme), this single-strand break site provides an additional amplification start site for the SD polymerase. This allows pre-amplification to continue at these sites without further primer hybridization.

[0033] For pre-amplification, an endonuclease is added to the SD polymerase. The endonuclease is preferably selected from the group consisting of Nt.Alwl, Nb.BbvCl, Nt.BbvCl, Nb.Bsml, Nt.BsmAl, Nt.BspQl, Nb.BsrDI, Nb.BssSI, Nt.BstNBI, Nb.Btsl and Nt.CviPll.

[0034] SD polymerase can, for example, be an enzyme mutant of the DNA polymerase of Bacillus subtilis phage Phi29, particularly EquiPhi29, or of Thermococcus litoralis, particularly Vent (exo-). The SD polymerase Vent (exo-) has the advantage of being stable and processive up to temperatures of 100°C. Therefore, it can be added to the nucleic acid mixture before denaturation without being damaged by the high denaturation temperature. However, these SD polymerases do not exhibit RNA processivity.

[0035] Preferably, the nucleic acid mixture is a mixture containing both DNA and RNA, thus utilizing the universal pre-amplification reaction provided by the process. The SD polymerase is preferably an enzyme mutant of the DNA polymerase of Bacillus stearothermophilus. These SD polymerases exhibit processivity not only towards DNA but also towards RNA. Particularly preferred enzyme mutants of the DNA polymerase of Bacillus stearothermophilus are selected from the group consisting of Bst, Bst 2.0, and Bst 3.0. Among these enzyme mutants, Bst 3.0 exhibits the highest RNA processivity, which is why Bst 3.0 is especially preferred.

[0036] In the case that SD polymerases are used which have 3'→5' exonuclease activity, the primers can be modified with 3'-terminal phosphorothiorate (PTO) groups and thus protected.

[0037] Even though SD polymerase is capable of pre-amplifying RNA in addition to DNA, it is preferable to use at least one reverse transcriptase during isothermal pre-amplification. In particular, RTx or SSIV are suitable. However, in principle, any reverse transcriptase that processes under similar reaction conditions to the SD polymerase used is conceivable. This specifically supports the pre-amplification of RNA, thus further shortening the required pre-amplification time. If an SD polymerase is used that lacks RNA processivity, then the use of a reverse transcriptase is necessary to process RNA in addition to DNA.

[0038] Since the method enables isothermal pre-amplification in a single reaction chamber where denaturation, hybridization, and isothermal pre-amplification take place, it can be easily integrated into microfluidic devices or lab-on-a-chip systems, thus enabling automated point-of-care analysis without additional equipment. For this purpose, a microfluidic device is provided, which includes an element for receiving a patient sample. An optional second element can be used to purify the nucleic acid mixture from the patient sample and provide it in its purest form for pre-amplification. A further element for pre-amplifying a nucleic acid mixture in the patient sample is configured to perform the pre-amplification using the method. This element can preferably contain pre-amplification reagents, particularly in a dry state as a bead.Pre-amplification can be performed directly with the patient sample without a prior purification step, or with the purified nucleic acid mixture. Finally, a component for detecting nucleic acid biomarkers in the pre-amplified nucleic acid mixture is included. Target detection can be achieved, for example, by adding specific primers and probes via isothermal amplification. Alternatively, the pre-amplified nucleic acids can be diluted and divided into individual wells to enable the simultaneous and individual detection of multiple targets. Detection methods using qPCR, CRISPR / Cas, NGS, or microarrays can also be employed for target detection.

[0039] Another object of the present invention is the use of chemically modified primers with random sequence featuring a SAMRS modification for the hybridization of nucleic acids. Brief description of the drawings

[0040] Exemplary embodiments of the invention are shown in the drawings and are explained in more detail in the following description. Fig. Figure 1 schematically shows a microfluidic device according to an embodiment of the invention. Fig. Figure 2 schematically shows the sequence of a method according to an embodiment of the invention. Fig. Figure 3 schematically shows the sequence of a procedure according to another embodiment of the invention. Fig. Figure 4 schematically shows the course of a process according to yet another embodiment of the invention. Fig. Figure 5 schematically shows the sequence of a procedure according to yet another embodiment of the invention. Fig. Figure 6 schematically shows a microfluidic device according to another embodiment of the invention. Fig. Figure 7 schematically shows the course of a process according to yet another embodiment of the invention. Fig. Figure 8 schematically shows the sequence of a procedure according to yet another embodiment of the invention. Fig. Figure 9 schematically shows the bonding of unmodified and SAMRS-modified nucleobases to each other. Exemplary embodiments of the invention

[0041] In one embodiment of the invention, a microfluidic device is provided, for example, as a disposable cartridge for an analytical device. Such cartridges comprise a fluidic layer, a pneumatic layer, and an elastomeric membrane arranged between the fluidic and pneumatic layers. The fluidic and pneumatic layers each have a substrate, for example, made of polycarbonate. The elastomeric membrane consists, for example, of thermoplastic polyurethane. Channels and chambers run through the fluidic layer, designed to transport reaction fluids and reaction mixtures in molecular diagnostics. Chemicals for carrying out chemical reactions are also located upstream of the fluidic layer. The pneumatic layer has channels that open onto the elastomeric membrane and have openings on an outer surface of the pneumatic layer.These openings allow the channels to be connected to a pneumatic manifold of the analyzer. When an overpressure is generated in one of the individually controllable channels of the pneumatic layer via the pneumatic manifold, the elastomer membrane is deflected into the fluidic layer. Conversely, when a negative pressure is generated in the channel, the elastomer membrane is deflected into the pneumatic layer. In this way, it is possible to manipulate fluid flows within the fluidic layer.

[0042] The representation in Fig. Figure 1 shows elements of the fluidic layer of the microfluidic device 10. A first element 11 is configured to receive a patient sample. For this purpose, it has an opening on the outside of the fluidic layer through which the patient sample can be introduced and absorbed, for example, onto silica gel in the first element 11. This first element 11 is fluidically connected to a second element 12 for purifying the patient sample. The second element 12 is fluidically connected to a third element 13 for pre-amplification of a nucleic acid mixture contained in the patient sample. The third element 13 is fluidically connected to a fourth element 14 for purifying the pre-amplification product. This fourth element 14 is fluidically connected, for example, to a fifth element 15 (see Figure 1). Fig. 6) for the detection of nucleic acid biomarkers in the purified pre-amplified nucleic acid mixture.

[0043] Fig. Figure 2 shows process steps 21-24, which in a first embodiment of the process according to the invention take place in a microfluidic device 10. The nucleic acid mixture 30 contains DNA 31 and RNA 32 as nucleic acids, which have secondary and tertiary structures. This nucleic acid mixture 30 is eluted in the first element 11 by means of an aqueous solution of chemically modified primers with random sequence 41, which have a ZNA modification and a SAMRS modification, and pumped into the second element 12.

[0044] Alternatively, the primers 41 mentioned are supplied as a lyophilisate on the cartridge and are dissolved with the aqueous solution before or after the elution of the nucleic acid mixture 30.

[0045] The primers 41 mentioned above can alternatively be placed upstream in the lyophilisate of the components required for pre-amplification.

[0046] In the second element 12, the first process step 21 involves denaturing the nucleic acid mixture 30 by heating the solution to 95°C for one minute. This yields single-stranded DNA 31 and single-stranded RNA 32, which are present in the solution with the chemically modified primers with random sequence 41, including ZNA and SAMRS modifications. The primers with random sequence 41 are, for example, chemically modified primers with three spermine groups attached to an N9 oligo (random sequence of 9 bases) (N9-ZNA3). Furthermore, three of the nine bases, for example, exhibit a SAMRS modification.

[0047] In the next process step 22, the single-stranded DNA 31 and the single-stranded RNA 32 are hybridized with the chemically modified primers with random sequence 41, featuring ZNA and SAMRS modifications. For this purpose, the solution is cooled to a temperature of, for example, 40°C and held at this temperature for one minute. This yields primer-hybridized DNA 51 and primer-hybridized RNA 52. In the next process step 23, the hybridized DNA 51 and the hybridized RNA 52 are pre-amplified. For this, a solution is first added from a Fig. In one chamber (not shown), another reaction solution is added and homogeneously mixed with the solution of hybridized DNA 51 and hybridized RNA 52. This additional reaction solution contains, for example, SD polymerase 61 designated Bst 3.0, as well as dNTPs, SSBs (single-stranded binding proteins), and a buffer. The homogeneous reaction mixture is then heated to a temperature of 50°C for, for example, 30 minutes. During this time, SD polymerase 61 performs pre-amplification of the sequences hybridized by the chemically modified primers with random sequence 41 (ZNA and SAMRS modifications) by strand displacement on both the hybridized DNA 51 and the hybridized RNA 52.

[0048] A detection reaction 24 then follows. For this, the reaction mixture is pumped into the third element 13. There, it is distributed, for example, across several cavities of a microarray and subjected to a qPCR reaction as a detection method. The third element 13 has a window on its side facing away from the elastomer membrane, which is oriented towards a sensor of the analysis device. This allows for the evaluation of the reactions that took place in the cavities of the microarray using fluorescence spectroscopy.

[0049] In an alternative embodiment of the first embodiment, the chemically modified primers with random sequence 41 have a SAMRS modification but no ZNA modification.

[0050] In a second embodiment of the method according to the invention, which is described in Fig. As shown in Figure 3, the reaction solution, which is mixed with the mixture of hybridized DNA 51 and hybridized RNA 52 in the second element 12, contains, in addition to the components provided in the first embodiment, a reverse transcriptase 62. This reverse transcriptase is, for example, RTx. In the isothermal pre-amplification step 23, the pre-amplification of the hybridized DNA 51 is carried out by the SD polymerase 61, as in the first embodiment. While the SD polymerase 61 also pre-amplifies individual hybridized RNA strands 52, as in the first embodiment, the pre-amplification of the hybridized RNA strands 52 in the second embodiment is additionally carried out after reverse transcription to cDNA by the reverse transcriptase 62. The further process steps of the second embodiment of the process are identical to those of the first embodiment.

[0051] While in the first two embodiments the pre-amplification 23 is performed as uMDA, in further embodiments the method according to the invention also enables an ssMDA: In a particularly advantageous third embodiment of the method according to the invention, which is described in Fig. As shown in Figure 4, the elution solution introduced into the first element 11 contains, in addition to chemically modified primers with random sequence 41 with ZNA modification and with SAMRS modification, also modified primers with specific sequence 42 and SAMRS modification. The modified primers with specific sequence 42 exhibit sequences that enable hybridization of DNA 31 and RNA 32 sequences for which SD polymerase 61 has only low processivity, or for sequences that are the target of subsequent investigations. While SD polymerase amplifies most DNA 31 and RNA 32 sequences a thousand times using, for example, the chemically modified primers with random sequence 41 with ZNA modification and SAMRS modification, these nucleic acids may also contain sequences that are only amplified twenty times.It is then intended that the primers with specific sequence 42 and SAMRS modification are primers with sequences that are specific for these weakly amplifiable DNA and RNA sequences. During hybridization 22, hybridized DNA strands 51 are formed, which carry only chemically modified primers with random sequence 41 with ZNA modification and SAMRS modification, and hybridized RNA strands 52, which carry only chemically modified primers with random sequence 41 with ZNA modification and SAMRS modification. In addition, hybridized DNA strands 53 are also formed, which carry chemically modified primers with random sequence 41 with ZNA modification and SAMRS modification, and chemically modified primers with specific sequence 42 with SAMRS modification.In addition, hybridized RNA strands 54 are formed, which carry chemically modified primers with random sequence 41 with ZNA modification and SAMRS modification and modified primers with specific sequence 42 with SAMRS modification.

[0052] Furthermore, hybridized DNA strands or RNA strands (each not in Fig. 4) form, which carry only chemically modified primers with specific sequence 42 with SAMRS modification. In pre-amplification 23, the SD polymerase 61 forms from the hybridized DNA strands 51 and RNA strands 52, which carry only chemically modified primers with random sequence 41 with ZNA modification and SAMRS modification, the same pre-amplification products 71 as can also be obtained in the first two embodiments of the process according to the invention. In addition, SD polymerase 61 forms a second amplification product 72, which has an increased proportion of the difficult-to-amplify sequences or target sequences, by pre-amplification of the hybridized DNA strands 53 and RNA strands 54, which carry chemically modified primers with random sequence 41 with ZNA modification and SAMRS modification and chemically modified primers with specific sequence 42 with SAMRS modification.Otherwise, the procedure according to the third embodiment of the invention corresponds to the uMDA according to the first embodiment of the invention.

[0053] In an alternative particularly advantageous embodiment of the third embodiment, the chemically modified primers with specific sequence 42 have, in addition to the SAMRS modification, also a ZNA modification.

[0054] In a further alternative particularly advantageous embodiment of the third embodiment, the chemically modified primers with random sequence 41 have only a SAMRS modification and the chemically modified primers with specific sequence 42 have a SAMRS modification and a ZNA modification.

[0055] Furthermore, an embodiment is advantageous in which both the chemically modified primers with random sequence 41 and the chemically modified primers with specific sequence 42 have a SAMRS modification, but no ZNA modification.

[0056] Further alternative embodiments are conceivable, wherein the chemically modified primers with specific sequence 42 have no modification and the chemically modified primers with random sequence 41 have a SAMRS modification or a ZNA and a SAMRS modification. Furthermore, the chemically modified primers with random sequence 41 can have a SAMRS modification or a SAMRS and a ZNA modification, and the chemically modified primers with specific sequence 42 can have a ZNA modification.

[0057] In a fourth embodiment of the invention, which is described in Fig. As shown in Figure 5, the third embodiment (in any embodiment) is modified such that the reaction solution, which is mixed with the hybridized nucleic acids 51-54 in the second element 12, additionally contains a reverse transcriptase 62, as in the second embodiment of the process. During pre-amplification 23, the hybridized DNA strands 51, 53 are then pre-amplified exclusively by the SD polymerase 61, while the hybridized RNA strands 52, 54 are also partially pre-amplified by the SD polymerase 61, but predominantly pre-amplified after reverse transcription to cDNA by the reverse transcriptase 62.

[0058] Otherwise, the sequence of operations of the fourth embodiment of the method according to the invention corresponds to the sequence of operations of the third embodiment.

[0059] Fig. Figure 6 shows elements of the fluidic layer of another embodiment of the microfluidic device 10. In contrast to the one in Fig. In the apparatus shown in Figure 1, a fifth element 15 is arranged between the third element 13 and the fourth element 14 for purifying the pre-amplification product. This fifth element 15 is configured for streptavidin-based purification to bring the relevant biomarker loci into a particularly favorable ratio compared to the less relevant loci. This can increase the sensitivity and specificity in downstream detection methods in the third element 13.

[0060] Using this device, the third and fourth embodiments of the method are modified into fifth and sixth embodiments. The modified primers with specific sequence 42, in addition to their corresponding modifications (depending on the embodiment), have sequences selected to bind, for example, to or adjacent with biomarker-relevant loci for viruses, bacteria, fungi, parasites, and tumors, for whose detection the microfluidic device 10 is intended. These modified primers with specific sequence 42, for example, have biotin as a selection marker at the 5' end. The second amplification product 72 is specific for these sequences in these modified embodiments.After completion of the pre-amplification 23 and before the subsequent detection step 24, an intermediate streptavidin-based purification is provided in the fifth element 15 in order to detect the associated biomarkers in the detection step 24 with high sensitivity and specificity.

[0061] Further modifications of the third and fourth embodiments to a seventh and eighth embodiment of the invention are described in the Fig. 7 and Fig. Figure 8 illustrates this. The modified primers with specific sequence 42, in addition to their corresponding modifications (depending on the embodiment), each have, for example, a 5'...GGATCNNNNN...3' sequence as a single-strand break site. In the third element 13, Nt.Alwl is added as endonuclease 13. After several pre-amplification cycles, the second pre-amplification product 72 is present in double-stranded form 73. The endonuclease 63 binds to the single-strand break sites and introduces a single-strand break. This break is recognized by the SD polymerase 62, which uses it as a target for further pre-amplification. If the single strand thus obtained becomes double-stranded again, the endonuclease 63 recognizes the single-strand break site on the same molecule once more, and the process is repeated.Thus, the yield of the second pre-amplification product 72 can be increased even further compared to the third and fourth embodiments.

[0062] The result of the detection reactions, which take place in the fourth element 14, is ultimately output via a user interface on the analysis device in all embodiments of the method according to the invention. The microfluidic device 10 can be disposed of as a single-use item after its use.

[0063] In Fig. Figure 9 shows the complementary nucleobases adenine (A) and thymine (T), linked by hydrogen bonds, as well as the complementary nucleobases guanine (G) and cytosine (C), linked by hydrogen bonds, in their natural form (without asterisks) or with an example of a SAMRS modification (with asterisks). Fig.As can be seen in section 9, SAMRS-modified nucleobases can bind more firmly or better to their respective complementary natural, non-SAMRS-modified nucleobases than to other SAMRS-modified nucleobases.

[0064] The natural nucleic bases G and C form three hydrogen bonds with each other. 2'-Deoxyinosine (G*) and N4-Ethyl-2'-Deoxycytidine (C*) bind to C and G via two hydrogen bonds each. G* and C*, however, form only one hydrogen bond with each other.

[0065] The natural nucleic bases A and T form two hydrogen bonds with each other. 2-Aminopurine-2'-deoxyriboside (A*) and 2'-deoxy-2-thiothymidine (T*) bind to T and A via two hydrogen bonds each. A* and T*, however, can only form one hydrogen bond with each other.

[0066] This shows that SAMRS-modified primers do not bind to each other in a nucleic acid mixture because they can only interact weakly. Such modified primers preferentially bind to non-SAMRS-modified nucleobases of the target DNA or RNA. QUOTES INCLUDED IN THE DESCRIPTION

[0000] This list of documents cited by the applicant was automatically generated and is included solely for the reader's convenience. The list is not part of the German patent or utility model application. The DPMA accepts no liability for any errors or omissions. Cited patent literature

[0000] DE 102022211087A1

[0005]

Claims

[1] Method for pre-amplification of a nucleic acid mixture (30) comprising the following steps: - Denaturing (21) the nucleic acid mixture (30) to obtain single-stranded nucleic acids (31, 32), - Hybridization (22) of the single-stranded nucleic acids (31, 32) with chemically modified primers with random sequence (41) wherein the chemically modified primers with random sequence (41) have a Self-Avoiding Molecular Recognition Systems (SAMRS) modification and - isothermal pre-amplification (23) of the hybridized nucleic acids (51-54), in particular as multiple displacement amplification (MDA), using a strand displacement (SD) polymerase (61). [2] Method according to claim 1, wherein, in addition to the chemically modified primers with random sequence (41), chemically modified primers with specific sequence (42) are also used for the hybridization (22) of the single-stranded nucleic acids (31, 32), and in particular, wherein the chemically modified primers with specific sequence (42) have a Self-Avoiding Molecular Recognition Systems (SAMRS) modification, [3] Method according to claim 1, characterized by , that the isothermal pre-amplification (23) takes place at a temperature in the range of 40°C to 72°C, and preferably in the range of 45°C to 65°C. [4] Method according to any one of the preceding claims, characterized by , that the primers with random sequence (41) and / or the primers with specific sequence (42) exhibit a Zip Nucleic Acids (ZNA) modification. [5] Method according to any one of the preceding claims, characterized by, that the primers with specific sequence (42) each have at least one selection marker. [6] Method according to any one of the preceding claims, characterized by that the primers (41, 42) have a single-strand break interface for an endonuclease (63). [7] Method according to any one of the preceding claims, characterized by , that the nucleic acid mixture (30) contains DNA (31) and RNA (32). [8] Method according to claim 7, characterized by , that the SD polymerase (61) is an enzyme mutant of the DNA polymerase of Bacillus stearothermophilus, in particular Bst 3.

0. [9] Method according to one of claims 7 or 8, characterized by , that in isothermal pre-amplification (23) at least one additional reverse transcriptase (62) is used. [10] Microfluidic device (10) comprising an element (11) for receiving a patient sample, an element (13) for pre-amplification of a nucleic acid mixture (30) in the patient sample and an element (14) for detection of nucleic acid biomarkers in the pre-amplified nucleic acid mixture (30), characterized by that the device (10) is set up to perform the pre-amplification (23) by means of a method according to any one of claims 1 to 9. [11] Use of chemically modified primers with random sequence (41) with a Self-Avoiding Molecular Recognition Systems (SAMRS) modification for nucleic acid hybridization.

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