Method and device for preamplifying a nucleic acid mixture

Abstract

The invention relates to a method for preamplifying a nucleic acid mixture (30). Said method comprises denaturing (21) the nucleic acid mixture (30) in order to obtain single-stranded nucleic acids (31, 32), hybridizing (22) the single-stranded nucleic acids (31, 32) with chemically modified primers (41, 42), and isothermally preamplifying (23) the hybridized nucleic acids (51, 52, 53, 54) by means of an SD polymerase (61). The invention also relates to a microfluidic device comprising: an element for receiving a patient sample; an element for preamplifying a nucleic acid mixture (20) in the patient sample; and an element for detecting nucleic acid biomarkers in the preamplified nucleic acid mixture (30). The device (10) is designed to carry out the preamplification (23) by means of the method.

Description

[0001] Description

[0002] title

[0003] Method and device for pre-amplification of a nucleic acid mixture

[0004] The present invention relates to a method for pre-amplification of a nucleic acid mixture. Furthermore, the present invention relates to a microfluidic device configured to perform pre-amplification using the method.

[0005] State of the art

[0006] In molecular diagnostics, diseases can be detected based on nucleic acids acting as biomarkers. These nucleic acids can be DNA or RNA. To detect as many pathogens as possible simultaneously based on such biomarkers, probe-based methods or sequencing can be used. However, if the sample input is small, these methods must be preceded by a pre-amplification of, for example, the relevant biomarkers or the entire mixture. However, simultaneous pre-amplification of DNA and RNA in a single reaction is not possible.

[0007] Pre-amplification can be performed using thermophilic enzymes, which require cyclic temperature changes. This results in increased reaction times, making such pre-amplification reactions unsuitable for point-of-care use. Isothermal amplification reactions are possible using mesophilic enzymes. However, these are less processive than thermophilic enzymes, so their use also results in very long reaction times.

[0008] Disclosure of the Invention The 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. The hybridized nucleic acids thus obtained are subjected to isothermal pre-amplification using an SD polymerase (strand displacement). SD polymerase is a DNA polymerase suitable for isothermal amplification reactions and is already known for its high strand displacement activity in amplification reactions. The method is based on the finding that SD polymerases are also suitable for pre-amplification reactions. The use of SD polymerase has several advantages. It enables isothermal pre-amplification without exhibiting the low processivity of mesophilic enzymes.In addition, 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 or no adaptation to different pathogens or biomarkers to be detected, as it can universally amplify DNA and RNA regardless of the application.

[0009] Before denaturing, the nucleic acid mixture can be detached, in particular, from a solid phase, for example silicon dioxide, using water or an elution buffer. Denaturing is preferably carried out by heating the nucleic acid mixture dissolved in water, with the heating being particularly preferably carried out to a temperature in the range of 75°C to 98°C and very particularly 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, particularly preferably for a period in the range of 30 seconds to 120 seconds. This reliably resolves secondary and tertiary structures of the nucleic acids. The chemically modified primers for hybridizing the single-stranded nucleic acids can preferably be brought into contact with the nucleic acid mixture prior to denaturing, particularly preferably by dissolving them in the water or elution buffer.They are insensitive to denaturation conditions. 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, the primers can anneal 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.

[0010] To prepare for isothermal pre-amplification, the solution of hybridized nucleic acids is mixed with SD polymerase. Furthermore, deoxynucleoside triphosphates (dNTPs) and buffer components can be added. The deoxynucleoside triphosphates serve as precursor building blocks for nucleic acids in the pre-amplification. The buffer components serve to adjust the pH value optimal for the pre-amplification. Furthermore, it is preferable to add single-strand binding proteins (SSBs). These single-strand binding proteins are proteins that bind to single-stranded DNA to facilitate isothermal pre-amplification by supporting the SD activity of the polymerase.

[0011] Isothermal pre-amplification is preferably not started until 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 within a range of 40°C to 72°C. A preferred pre-amplification period is in the range of 10 minutes to 120 minutes. The higher the temperature during pre-amplification, the faster it proceeds and, usually, the shorter the pre-amplification period can be advantageously selected. Compared to pre-amplifications with mesophilic enzymes at 40°C or lower, this can significantly shorten the reaction time. The reaction is accelerated particularly at a temperature in the range of 45°C to 65°C.The method also has the advantage that it can be used for pre-amplification of RNA even at such high temperatures, whereas other methods only allow pre-amplification of RNA at lower temperatures.

[0012] The primers are preferably primers that have at least one 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. ZNA primers (Zip Nucleic Acids) are particularly preferred. These are spermine-modified primers. These 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 extension of the primers. Furthermore, with appropriate spermine loading of the primers, they reduce self-hybridization of the primers.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.

[0013] In a preferred embodiment of the method, the primers are modified primers with a random sequence (random primers). These primers, which in particular have a defined chain length, preferably between 6 and 15 bases, and consist of a purely random sequence, have the advantage of covering all regions of the nucleic acid template to be amplified with a high probability. They enable the isothermal pre-amplification to be carried out as uMDA (universal multiple displacement amplification).

[0014] In a further embodiment of the method, it is preferred that, in addition to the modified primers with a random sequence, primers with a specific sequence are used to hybridize the single-stranded nucleic acids. The additional use of the specific primers enables ssMDA (semi-specific multiple displacement amplification). These primers with a specific sequence can also carry modifications that increase the processivity of SD polymerase. In particular, these are spermine groups. ZNA primers with a specific sequence are particularly preferred, as they increase the processivity of SD polymerase and, when appropriately loaded, have a repulsive effect on each other. This modification helps prevent unwanted interactions between the primers with a specific sequence. Furthermore, it allows the use of a large number of different primers with a specific sequence.

[0015] Sequence-specific primers are defined as primers that not only have a defined chain length but also a defined sequence. This sequence can be selected to bind to specific loci on the nucleic acids, for example, flanking one or more targets, thus favoring the enrichment of one or more targets relative to the background. Alternatively or additionally, the sequence can be selected to bind to nucleic acid sequences for which SD polymerase exhibits lower processivity compared to other nucleic acid sequences, for example, due to difficult sequences with a high GC content. By enhancing the pre-amplification of otherwise insufficiently amplified sequences, not only universal but also more uniform pre-amplification is enabled.

[0016] The primers with a specific sequence preferably contain at least one selectable marker, such as biotin. This selectable marker is advantageous when the primers are specific for a target and are 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 selectable markers.

[0017] It is preferable for both modified primers with random sequences and primers with specific sequences that the primers contain a single-strand break site. This single-strand break site provides an additional amplification starting point for SD polymerase after the single-strand break is created by a suitably added endonuclease (nicking enzyme). This allows pre-amplification to continue at these sites without further primer hybridization, thus increasing processivity.

[0018] During pre-amplification, an endonuclease is added to the SD polymerase. The endonuclease is preferably selected from the group consisting of Nt.AlwI, Nb.BbvCI, Nt.BbvCI, Nb.Bsml, Nt.BsmAI, Nt.BspQI, Nb.BsrDI, Nb.BssSI, Nt.BstNBI, Nb.BtsI, and Nt.CviPII.

[0019] The SD polymerase can, for example, be an enzyme mutant of the DNA polymerase of Bacillus subtilis phage Phi29, especially EquiPhi29, or of Thermococcus litoralis, especially Vent (exo-). 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 lack RNA processivity.

[0020] The nucleic acid mixture is preferably a mixture containing both DNA and RNA, thus exploiting the possibility of a universal pre-amplification reaction provided by the method. The SD polymerase is preferably an enzyme mutant of the DNA polymerase of Bacillus stearothermophilus. These SD polymerases exhibit not only processivity towards DNA but also processivity 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 particularly preferred.

[0021] If SD polymerases with 3'->5' exonuclease activity are used, the primers can be modified with 3'-terminal phosphorothioate groups (PTO) and thus protected. Even if the SD polymerase is capable of pre-amplifying RNA as well as DNA, it is preferable to use at least one reverse transcriptase during isothermal pre-amplification. This is particularly true for RTx or SSIV. In principle, however, all reverse transcriptases that process under similar reaction conditions to the SD polymerase used are conceivable. This specifically supports the pre-amplification of RNA, so that the required pre-amplification time can be shortened even further. If an SD polymerase is used that lacks RNA processivity, then the use of a reverse transcriptase is necessary to process RNA as well as DNA.

[0022] Since the method enables isothermal pre-amplification in a single reaction chamber in which denaturation, hybridization, and isothermal pre-amplification take place, it can be easily integrated into microfluidic devices or lab-on-chip systems, enabling automated analysis at the point of care without additional equipment. For this purpose, a microfluidic device is provided which has an element for receiving a patient sample. Another element can optionally be used to purify the nucleic acid mixture from the patient sample and make it available for pre-amplification in its purest form. Another element for pre-amplification of a nucleic acid mixture in the patient sample is designed to carry out the pre-amplification using the method. Reagents for the pre-amplification can preferably be stored upstream in this element, particularly in a dry state as a bead.Pre-amplification can be performed directly with the patient sample without a prior purification element or with the purified nucleic acid mixture. Finally, an element for detecting nucleic acid biomarkers is provided in the pre-amplified nucleic acid mixture. Target detection can be achieved via isothermal amplification, for example, by adding specific primers and probes. However, the pre-amplified nucleic acids can also be diluted and distributed into individual wells to detect multiple targets simultaneously and individually. Detection methods using qPCR, CRISPR / Cas, NGS, or microarrays can also be used for target detection. Brief description of the drawings.

[0023] Embodiments of the invention are illustrated in the drawings and are explained in more detail in the following description.

[0024] Figure 1 schematically shows a microfluidic device according to an embodiment of the invention.

[0025] Figure 2 shows schematically the sequence of a method according to an embodiment of the invention.

[0026] Figure 3 shows schematically the sequence of a method according to another embodiment of the invention.

[0027] Figure 4 shows schematically the course of a method according to yet another embodiment of the invention.

[0028] Figure 5 shows schematically the sequence of a method according to yet another embodiment of the invention.

[0029] Figure 6 schematically shows a microfluidic device according to another embodiment of the invention.

[0030] Figure 7 shows schematically the course of a method according to yet another embodiment of the invention.

[0031] Figure 8 shows schematically the sequence of a method according to yet another embodiment of the invention.

[0032] Embodiments of the invention

[0033] In one embodiment of the invention, a microfluidic device is provided, which is designed, for example, as a disposable cartridge for an analysis device. Such cartridges have a fluidic layer, a pneumatic layer, and an elastomer membrane arranged between the fluidic layer and the pneumatic layer. The fluidic layer and the pneumatic layer each have a substrate made, for example, of polycarbonate. The elastomer membrane is made, for example, of thermoplastic polyurethane. Channels and chambers run within the fluidic layer and are intended to transport reaction liquids and reaction mixtures in molecular diagnostics. Furthermore, chemicals for carrying out chemical reactions are arranged upstream of the fluidic layer. The pneumatic layer has channels that open at the elastomer membrane and have openings on an outer side of the pneumatic layer.These openings allow the channels to be connected to a pneumatic manifold of the analysis device. When positive pressure is generated in one of the individually controllable channels of the pneumatic layer using the pneumatic manifold, the elastomer membrane is deflected into the fluidic layer. Conversely, if negative pressure is generated in the channel, the elastomer membrane is deflected into the pneumatic layer. This makes it possible to manipulate fluid flows in the fluidic layer.

[0034] The illustration in 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 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-amplifying 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 to a fifth element 15 for detecting nucleic acid biomarkers in the purified, pre-amplified nucleic acid mixture.

[0035] Figure 2 shows process steps 21-24, which, in a first embodiment of the method 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 using an aqueous solution of ZNA primers and pumped into the second element 12. There, 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 ZNA primers 41. For example, the ZNA primers 41 are modified primers with three spermine groups on an N9 oligo (random sequence of 9 bases) (N9-ZNA3).The next process step 22 involves hybridizing the single-stranded DNA 31 and the single-stranded RNA 32 with the ZNA primers 41. For this purpose, the solution is cooled to a temperature of 40°C, for example, and held at this temperature for one minute. This produces primer-hybridized DNA 51 and primer-hybridized RNA 52. In the next process step 23, a pre-amplification of the hybridized DNA 51 and the hybridized RNA 52 is carried out. For this purpose, a further reaction solution is first added to the solution from a chamber not shown in Figure 1 and homogeneously mixed with the solution of the hybridized DNA 51 and hybridized RNA 52. The further reaction solution contains, for example, an SD polymerase 61 called 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 30 minutes, for example.SD polymerase 61 performs a pre-amplification of the sequences hybridized using the ZNA primers 41 by strand displacement on both the hybridized DNA 51 and the hybridized RNA 52. This is followed by a detection reaction 24. For this purpose, the reaction mixture is pumped into the third element 13. There, it is distributed, for example, among several wells 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 faces a sensor of the analysis device. This enables evaluation of the reactions taking place in the wells of the microarray using fluorescence spectroscopy.In a second embodiment of the method according to the invention, the reaction solution, which is mixed in the second element 12 with the mixture of hybridized DNA 51 and hybridized RNA 52, contains, in addition to the components provided in the first embodiment, a reverse transcriptase 62. This is, for example, RTx. In the isothermal pre-amplification step 23, the pre-amplification of the hybridized DNA 51 takes place, as in the first embodiment, by the SD polymerase 61. 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 takes place after reverse transcription to cDNA by means of the reverse transcriptase 62. The further method steps of the second embodiment of the method correspond to the first embodiment.

[0036] While in the first two embodiments the pre-amplification 23 is carried out as uMDA, the method according to the invention also enables ssMDA in further embodiments:

[0037] In a third embodiment of the method according to the invention, which is shown in Figure 4, the elution solution introduced into the first element 11 contains, in addition to the modified ZNA primers 41 with a random sequence, also modified primers 42 with a specific sequence. The modified primers 42 with a specific sequence have sequences that enable hybridization of DNA 31 and RNA 32 sequences for which SD polymerase 61 has only low processivity. While SD polymerase amplifies most DNA 31 and RNA 32 sequences a thousand times, for example, using the modified ZNA primers 41 with a random sequence, these nucleic acids can also have sequences that are amplified only twenty times.It is then intended that the modified ZNA primers 42 with a specific sequence are primers with sequences that are specific for these weakly amplifiable DNA sequences and RNA sequences. During hybridization 22, as in the first two embodiments, hybridized DNA strands 51 are formed, which carry only modified ZNA primers 41 with a random sequence, and hybridized RNA strands 52 are formed, which carry only ZNA primers 41 with a random sequence. In addition, hybridized DNA strands 53 are formed, which carry modified ZNA primers 41 with a random sequence and modified primers 42 with a specific sequence. Furthermore, hybridized RNA strands 54 are formed, which carry modified ZNA primers 41 with a random sequence and modified primers 42 with a specific sequence.During pre-amplification 23, SD polymerase 61 forms the same pre-amplification products 71 from the hybridized DNA strands 51 and RNA strands 52, which carry only ZNA primers 41 with a random sequence, as can also be obtained in the first two embodiments of the method according to the invention. In addition, SD polymerase 61 forms a second amplification product 72, which has an increased proportion of difficult-to-amplify sequences, by pre-amplifying the hybridized DNA strands 53 and RNA strands 54, which carry modified ZNA primers 41 with a random sequence and modified primers 42 with a specific sequence, at the sequences to which the modified primers 42 with a specific sequence have annealed. Otherwise, the process of the method according to the third embodiment of the invention corresponds to the uMDA according to the first embodiment of the invention.

[0038] In a fourth embodiment of the invention, illustrated in Figure 5, the fourth embodiment is modified such that the reaction solution, which is mixed with the hybridized nucleic acids 51-54 in the second element 12, as in the second embodiment of the method, additionally contains a reverse transcriptase 62. 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 are predominantly pre-amplified after reverse transcription to cDNA by means of the reverse transcriptase 62. Otherwise, the sequence of the fifth embodiment of the method according to the invention corresponds to the sequence of the third embodiment. Figure 6 shows elements of the fluidic layer of another embodiment of the microfluidic device 10.In contrast to the device shown in Figure 1, a fifth element 15 for purifying the pre-amplification product is arranged between the third element 13 and the fourth element 14. 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.

[0039] When using this device, the third and fourth embodiments of the method are modified to form fifth and sixth embodiments. The modified primers 42 with a specific sequence have sequences selected such that they bind, for example, to or adjacent to biomarker-relevant loci for viruses, bacteria, fungi, and tumors, for whose detection the microfluidic device 10 is intended. These modified primers 42 with a specific sequence have, for example, 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.

[0040] Further modifications of the third and fourth embodiments to a seventh and eighth embodiment of the invention are shown in Figures 7 and 8. Here, it is provided that the modified primers 42 with a specific sequence each have, for example, a 5'...GGATCNNNNN...3' sequence as a single-strand break cleavage site. In the third element 13, Nt.Alwl is added as endonuclease 13. After the second pre-amplification product 72 is present in double-stranded form after several rounds of pre-amplification, endonuclease 63 binds to the single-strand break cleavage site and inserts a single-strand break. This cleavage is recognized by SD polymerase 62, which uses it as a point of attack for further pre-amplification. If the resulting single strand becomes double-stranded again, endonuclease 63 again recognizes the single-strand break cleavage site on the same molecule, and the process repeats.Thus, the yield of the second pre-amplification product 72 can be increased even further compared to the third and fourth embodiments.

[0041] The result of the detection reactions taking place in the fourth element 14 is finally output in all embodiments of the method according to the invention via a user interface on the analysis device. The microfluidic device 10 can be disposed of as a disposable item after use.

Claims

Claims 1. A method for pre-amplifying 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 (41), and isothermal pre-amplification (23) of the hybridized nucleic acids (51-54) using an SD polymerase (61).

2. Method according to claim 1, characterized in that the isothermal pre-amplification (23) takes place at a temperature in the range of 40°C to 72°C.

3. Method according to claim 2, characterized in that the isothermal pre-amplification (23) takes place at a temperature in the range of 45°C to 65°C.

4. Method according to one of claims 1 to 3, characterized in that the primers (41) are ZNA primers.

5. Method according to one of claims 1 to 4, characterized in that the primers (41) are modified primers with a random sequence.

6. Method according to one of claims 1 to 5, characterized in that a hybridization (22) of the single-stranded nucleic acids (31, 32) with primers (42) having a specific sequence is also carried out.

7. The method according to claim 6, characterized in that the primers with specific sequence (42) each have at least one selection marker. Method according to one of claims 1 to 7, characterized in that the primers (41, 42) have a single-strand break cleavage site for an endonuclease (63). Method according to one of claims 1 to 8, characterized in that the nucleic acid mixture (30) contains DNA (31) and RNA (32). Method according to claim 9, characterized in that the SD polymerase (61) is an enzyme mutant of the DNA polymerase of Bacillus stearothermophilus. Method according to claim 10, characterized in that the SD polymerase (61) is Bst 3.

0. Method according to one of claims 9 to 11, characterized in that at least one reverse transcriptase (62) is additionally used in the isothermal pre-amplification (23).Microfluidic device (10) comprising an element (11) for receiving a patient sample, an element (13) for pre-amplifying a nucleic acid mixture (30) in the patient sample and an element (14) for detecting nucleic acid biomarkers in the pre-amplified nucleic acid mixture (30), characterized in that the device (10) is configured to carry out the pre-amplification (23) by means of a method according to one of claims 1 to 12.

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