Method and kit for amplifying a target nucleic acid sequence

Abstract

The invention relates to a method for amplifying a target nucleic acid sequence from a starting material containing nucleic acids, the method comprising the steps of: (a) providing a reaction chamber in which the starting material and all reagents for the subsequent method steps (b) to (e) are present, wherein the reagents comprise a plurality of preamplification primers (50) and a preamplification polymerase (60), wherein the reagents comprise at least one pair of PCR primers (10, 110) and a PCR polymerase (70), wherein the starting material forms a reaction mixture together with the reagents; (b) heating the reaction mixture to 50 to 85°C; (c) preamplifying the nucleic acids by means of isothermal amplification using the preamplification primers (50) and the preamplification polymerase (60); (d) heating the reaction mixture to 90 to 100°C; (e) amplifying the target nucleic acid sequence by means of PCR using the PCR primers (10, 110) and the PCR polymerase (70).

Description

[0001] R.414809

[0002] Method and kit for amplifying a target nucleic acid sequence

[0003] The invention relates to a method for amplifying a target nucleic acid sequence from a starting material containing nucleic acids. The invention further relates to a kit for amplifying a target nucleic acid sequence from a starting material containing nucleic acids and to a use of the kit for amplifying a target nucleic acid sequence from a starting material containing nucleic acids.

[0004] The study of individual cells is of great importance in research and medicine, as it provides insights into the biology of individual cells and into the spatial and temporal regulation of cell clusters.

[0005] An important application of single-cell analysis is the examination of circulating tumor cells (CTCs). These are tumor cells that have detached from a solid primary tumor and circulate in the bloodstream. These cells can then enter tissues elsewhere from the blood and be responsible for the development of tumor metastases.

[0006] The quantification of CTCs can be used in certain types of cancer as a predictive marker for the further course of the disease or as a marker for the response to a specific cancer therapy.

[0007] To examine circulating tumor cells (CTCs), the cells are preferably first enriched or isolated using established methods. For single-cell analysis, the CTCs are then isolated. Their nucleic acids, for example, can then be analyzed for mutations and / or methylation patterns. For this purpose, the nucleic acids of the CTCs are subjected to sequencing, for example. Quantitative PCR can also be used to analyze the nucleic acids of the CTCs. Due to the small amount of nucleic acids available when working at the single-cell level, pre-amplification of the nucleic acids from the individual cells is usually necessary before amplification.

[0008] Two of the nucleic acids can be analyzed using PCR and, if necessary, subsequent sequencing. The information obtained from the analysis of the nucleic acids of the CTCs can be used, for example, to make a treatment decision.

[0009] German patent application DE 10 2022 211 087 A1 discloses a method for the pre-amplification of nucleic acids by means of isothermal amplification. For subsequent quantitative PCR, the reaction mixture is pumped into a new reaction chamber after pre-amplification, in which the PCR takes place.

[0010] Methods for the pre-amplification and subsequent amplification of nucleic acids are also known that do not require transferring the reaction mixture or the pre-amplified nucleic acids to a new reaction chamber for amplification as an intermediate step. In these methods, the pre-amplification intermediate step involves purification of the pre-amplified nucleic acids or buffering of the pre-amplification reaction mixture. Regardless of the type of intermediate step, the pre-amplified nucleic acids are only combined with the reagents required for the amplification of a target nucleic acid sequence during or after the intermediate step. This ensures that amplification only occurs after the pre-amplification has been completed. Furthermore, this method prevents the pre-amplification process from continuing during the amplification stage.After the intermediate step, the amplification of the target nucleic acid sequence is carried out, for example by PCR.

[0011] The chain of process steps, particularly the intermediate step, can lead to a loss of nucleic acids. This limits the quality of the work, especially when using a small amount of starting material, such as in single-cell studies.

[0012] There is a need to provide further methods for amplifying a target nucleic acid sequence.

[0013] A method for amplifying a target nucleic acid sequence from a starting material containing nucleic acids is presented. R.414809

[0014] 3

[0015] The method for amplifying a target nucleic acid sequence from a starting material containing nucleic acids includes:

[0016] (a) Providing a reaction chamber in which the starting material and all reagents for the subsequent process steps (b) to (e) are present, wherein the reagents comprise a plurality of pre-amplification primers and a pre-amplification polymerase, wherein the reagents comprise at least one pair of PCR primers and a PCR polymerase, wherein the starting material together with the reagents forms a reaction mixture,

[0017] (b) Heating the reaction mixture to 50 to 85°C,

[0018] (c) Pre-amplification of nucleic acids by isothermal amplification using pre-amplification primers and pre-amplification polymerase,

[0019] (d) Heating the reaction mixture to 90 to 100°C,

[0020] (e) Amplifying the target nucleic acid sequence by PCR using the PCR primers and the PCR polymerase, wherein the pre-amplification primers (50) are in an active form in step (a), wherein the pre-amplification polymerase (60) remains stable under the conditions in step (b) and is inactivated under the conditions in step (d), wherein the PCR primers (10, 110) and the PCR polymerase (70) in the step

[0021] (a) each in an inactive form, wherein the PCR primers (10, 110) and the PCR polymerase (70) are each under the conditions in the step

[0022] (b) remain inactive and can be activated under the conditions in step (d).

[0023] The method amplifies (replicates) a target nucleic acid sequence from a starting material containing nucleic acids. The nucleic acids in the starting material are a mixture of nucleic acids with different sequences. The nucleic acids in the starting material can include DNA and / or RNA. The method can be used, for example, to determine the presence of a specific target nucleic acid sequence in the starting material or to prepare the target nucleic acid sequence for subsequent sequencing. The method can also be used to determine the absence of R.414809

[0024] 4. To determine a specific target nucleic acid sequence, for example, in a diagnostic test. In this case, it is possible that the starting material does not contain the target nucleic acid sequence to be amplified. Therefore, the nucleic acids of the starting material may or may not include the target nucleic acid sequence to be amplified, depending on the specific question being addressed. In most applications of the method, the nucleic acids of the starting material do include the target nucleic acid sequence to be amplified. The target nucleic acid sequence can be in the form of DNA or in the form of RNA, for example, in the form of mRNA. The target nucleic acid sequence typically has a length of 50 to 1,000 nucleotides.

[0025] The process is an in-vitro / intra-tro process.

[0026] The method is suitable for small quantities of starting material. The starting material can be, for example, a single cell, especially a single tumor cell. The cell can be a living or a fixed cell.

[0027] The process takes place in a reaction chamber in which, at the start of the process, the starting material and all reagents for the subsequent process steps (b) to (e) are present. The reaction chamber, which can also be referred to as the reaction compartment, is typically a sealed space. Sealing the reaction chamber serves, in particular, to prevent evaporation of reagents, especially a buffer, during the process. The reaction chamber can be, for example, a sealed reaction vessel or a droplet. The reaction chamber can, in particular, be a cavity (also referred to as a reaction cavity) of a microcavity array. The microcavity array can be arranged in a microfluidic cartridge. The reaction chamber can be sealed, for example, by a lid or by covering it with oil.

[0028] The process includes providing the reaction chamber in which the starting material and all reagents for the subsequent process steps (b) to (e) are present (step (a)). Thus, all reagents required for the subsequent process steps are present in the reaction chamber from the outset. Therefore, it is not necessary to add reagents R.414809 during the process.

[0029] 5. Furthermore, no reagents need to be removed during the process. In fact, the reaction chamber can remain closed for the entire duration of the procedure. The process takes place in a single reaction chamber.

[0030] The procedure comprises two sequential molecular biological reactions: pre-amplification of the nucleic acids of the starting material as the first molecular biological reaction (hereinafter also referred to as the first reaction; step (c)) and amplification of the target nucleic acid sequence as the second molecular biological reaction (hereinafter also referred to as the second reaction; step (e)). The reagents for procedure steps (b) to (e) therefore include both reagents for the pre-amplification of the nucleic acids and reagents for the amplification of the target nucleic acid sequence. The pre-amplification of the nucleic acid is carried out by isothermal amplification. The reagents for this include, in particular, a plurality of pre-amplification primers and a pre-amplification polymerase. The subsequent amplification of the target nucleic acid sequence is carried out by PCR.The reagents required for this include, in particular, at least one pair of PCR primers and a PCR polymerase.

[0031] The pre-amplification and amplification processes occur consecutively.

[0032] During pre-amplification of the nucleic acids of the starting material, the pre-amplification primers and pre-amplification polymerase are active, while the PCR primers and PCR polymerase are inactive. The PCR primers and PCR polymerase are activated after pre-amplification and are active during amplification of the target nucleic acid sequence.

[0033] The term "conditions," as used here, refers to the specific circumstances under which a particular step of the procedure takes place. These specific circumstances include, for example, temperature, duration, and the concentration of the reagents. The term "conditions" refers primarily to the temperature used in a particular step and its duration, because these two parameters are most important for the relevant properties (such as stability, inactivity, activatability, etc.) of the reagents in question.

[0034] The term “conditions in step (b)” as used here refers in particular to those for heating the reaction mixture in R.414809

[0035] 6

[0036] The term "conditions in step (d)" refers in particular to the temperature used in the step, which may be from 50 to 85°C, preferably 60 to 85°C, and more preferably 70 to 80°C, and to the duration of the heating. Similarly, the term "conditions in step (d)" as used here refers in particular to the temperature used for heating the reaction mixture in the step, which may be from 90 to 100°C, preferably 90 to 98°C, and more preferably 93 to 95°C, and to the duration of the heating.

[0037] Pre-amplification primers contain primers with a random sequence. Such primers are also referred to as randomized primers or random primers. This ensures that all regions of the nucleic acids in the starting material are pre-amplified with a high probability. Pre-amplification primers can be exclusively randomized primers. Alternatively, in addition to the randomized primers, pre-amplification primers can also contain primers that are specific for the target nucleic acid sequence (target-specific primers).

[0038] The pre-amplification primers may have a chemical modification. For example, the pre-amplification primers may be LNA (locked nucleic acid) primers or ZNA (zipped nucleic acid) primers.

[0039] The pair of PCR primers is specific for the target nucleic acid sequence. In other words, the PCR primers are target-specific primers. The pair of PCR primers consists of a forward primer and a reverse primer. The PCR primers are complementary to the beginning and end regions of the target nucleic acid sequence, respectively.

[0040] PCR primers can be flanked primers. A flanked primer is a primer that has a target-specific region and a non-specific flank region. The target-specific region is complementary to a segment of the target nucleic acid sequence and can also be called the sequence-specific region. The non-specific flank region, on the other hand, is not complementary to the target nucleic acid sequence and can also be called the non-target-specific region. The target-specific region is located at the 3' end of the flanked primer. The non-specific flank region is located at the 5' end of the flanked primer. The non-specific flank region serves to introduce one or more additional sequence segments into the PCR products. An example of such an additional sequence segment is a barcode or identification code.

[0041] 7. Rung sequence. For each reaction chamber, an individual barcode or identification sequence can be used to enable subsequent assignment of the amplicates to the individual reaction chambers and thus to the respective starting material.

[0042] The pre-amplification primers are present in an active form in step (a). The term "active form," as used here, means that the reagents are immediately available for a reaction. With regard to the pre-amplification primers, this means that they are readily available for the first reaction, i.e., the pre-amplification of the nucleic acids. The pre-amplification primers are present in solution. Under the conditions in step (b), the pre-amplification primers remain active.

[0043] In step (a), the pre-amplification polymerase is present either in an active form or in an inactive form that can be activated under the conditions in step (b). The inactive form of the pre-amplification polymerase, which can be activated under the conditions in step (b), may, for example, exhibit a blockage of its polymerase activity by antibodies or aptamers. Under the conditions in step (b), these antibodies or aptamers detach from the pre-amplification polymerase, thus converting it into an active form. In this way, the non-specific incorporation of deoxynucleotide triphosphates (dNTPs) by the pre-amplification polymerase is prevented prior to step (b).

[0044] The pre-amplification polymerase is typically in solution. Alternatively, the pre-amplification polymerase can be immobilized on a surface. In this case, the linker connecting the immobilized pre-amplification polymerase to the surface is chosen such that the pre-amplification polymerase is in an active form or in an inactive form that can be activated under the conditions in step (b).

[0045] The pre-amplification polymerase remains stable under the conditions in step (b). Thus, the pre-amplification polymerase is available for nucleic acid pre-amplification. R.414809

[0046] 8

[0047] The pre-amplification polymerase is inactivated under the conditions in step (d). During the process, the pre-amplification polymerase is inactivated by appropriate heating of the reaction mixture (step (d)). This terminates the first reaction. Furthermore, the pre-amplification polymerase is then unavailable for the second reaction. In this way, the progression of the first reaction during the second reaction is prevented.

[0048] In step (a), the PCR primers and PCR polymerase are each in an inactive form. The term "inactive form," as used here, means that the reagents are not immediately available for a reaction. With regard to the PCR primers and PCR polymerase, this means that they are not available for the pre-amplification of the nucleic acids. The PCR primers and PCR polymerase remain inactive under the conditions in step (b) and can be activated under the conditions in step (d). Thus, the PCR primers and PCR polymerase are temperature-activated. During the procedure, the PCR primers and PCR polymerase are activated by appropriate heating of the reaction mixture (step (d)). The PCR primers and PCR polymerase are then available for the second reaction, that is, the amplification of the target nucleic acid sequence.The required heat activation of the PCR primers and the PCR polymerase ensures that the second reaction only takes place after the first reaction has been completed.

[0049] The inactive form of the PCR primers can be achieved, for example, by immobilizing them on a surface. In step (d), the immobilized and therefore inactive PCR primers are dissolved and thus activated by heating the reaction mixture.

[0050] The inactive form of the PCR primers can be achieved, for example, through chemical modification of the PCR primers. Such chemically modified PCR primers may be present in solution. In step (d), the chemical modification is reversed, for example, by heating the reaction mixture, such that the PCR primers assume an active form.

[0051] A combination of immobilization and chemical modification of the PCR primers is also possible to create an inactive form of the PCR primers. R.414809

[0052] 9

[0053] The inactive form of PCR polymerase can be achieved, for example, through chemical modification of the PCR polymerase, a mutation in the amino acid sequence of the PCR polymerase, blockage of the PCR polymerase by antibodies, or blockage of the PCR polymerase by aptamers. In each case, the inactive PCR polymerase is converted into an active form by heating the reaction mixture in step (d). For example, in the case of blockage of the PCR polymerase by antibodies or aptamers, the antibodies or aptamers separate from the PCR polymerase upon heating. The PCR polymerase remains stable under the conditions in step (d). Thus, the PCR polymerase is available for amplification of the target nucleic acid sequence.

[0054] Pre-amplification primers have a lower melting point compared to PCR primers. This is because the binding of the pre-amplification primers to the nucleic acids of the starting material occurs at a lower temperature (typically in the range of 4 to 50°C) than the binding of PCR primers during PCR (typically in the range of 55 to 68°C). The lower melting point can be achieved, for example, by using pre-amplification primers that are shorter than PCR primers or by chemically modifying the pre-amplification primers.

[0055] The reagents for the subsequent process steps (b) to (e) also include reagents required for both molecular biological reactions of the procedure. These include a suitable buffer compatible with the pre-amplification polymerase and the PCR polymerase, as well as nucleotides (dNTPs). These reagents are typically each present in an active form. They may also each be present in an inactive form that can be activated under the conditions in step (b).

[0056] The starting material, together with the reagents, forms a reaction mixture.

[0057] Step (a) is typically performed at a temperature of 4 to 45°C. Preferably, step (a) is performed at a temperature of 4 to 25°C. For example, step (a) is performed at a temperature of 20 to 25°C. R.414809

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[0059] After step (a), the reaction mixture is heated to 50 to 85°C (step (b)). Preferably, the reaction mixture is heated to 60 to 85°C, more preferably to 70 to 80°C, for example to 80°C. Step (b) is typically carried out for 1 to 30 min. Preferably, step (b) is carried out for 1 to 10 min, more preferably for 1 to 5 min. For example, step (b) is carried out for 30 min at 50°C or for 1 min at 80°C. The conditions in step (b) are selected, among other things, depending on the pre-amplification polymerase used. The pre-amplification polymerase must remain stable under the conditions in step (b) so that it is available for the subsequent pre-amplification of the nucleic acids.

[0060] If the starting material is one or more cells, thermal lysis of the cell(s) takes place in step (b). This releases the nucleic acids of the cell(s).

[0061] In step (b), heating leads to a partial breakdown of the base pairing in the nucleic acids of the starting material. This partial breakdown of the base pairing facilitates the initiation of the subsequent first reaction by making it easier for the pre-amplification primers to bind to complementary regions of the nucleic acids in the starting material. Complete denaturation of the double-stranded nucleic acids of the starting material into their single strands is not required for the initiation of the subsequent first reaction.

[0062] If the pre-amplification polymerase is in an inactive form that can be activated under the conditions in step (b), then in step (b) the pre-amplification polymerase is also activated.

[0063] After heating the reaction mixture to 50 to 85°C, the nucleic acids are pre-amplified by isothermal amplification using pre-amplification primers and pre-amplification polymerase (step (c)). Isothermal amplification is also known as isothermal amplification. Isothermal amplification takes place at a constant reaction temperature (isothermal). Isothermal amplification is a well-known amplification technique with various known R.414809

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[0065] Variants. Isothermal amplification can, for example, be strand displacement amplification (SDA) or loop-mediated isothermal amplification (LAMP).

[0066] Isothermal amplification is specifically a strand displacement amplification (SDA). SDA is also known as multiple displacement amplification (MDA). In SDA, a DNA polymerase with strand displacement activity, also called SD polymerase, displaces an existing second strand of double-stranded DNA while synthesizing a new DNA strand from the first strand, which acts as a template. The synthesis of the new DNA strand occurs when the DNA polymerase continuously adds single nucleotides to a primer bound to the template. The new DNA strand then has the same nucleotide sequence as the second strand. In this case, the primer is one of the pre-amplification primers, and the SD polymerase is the pre-amplification polymerase.In loop-mediated isothermal amplification (LAMP), an SD polymerase also serves as a pre-amplification polymerase. Examples of suitable SD polymerases are the Bst 3.0 DNA polymerase from New England Biolabs, Inc. (New England Biolabs, Inc., Ipswich, MA, USA), the Bst 2.0 DNA polymerase from New England Biolabs, Inc. (New England Biolabs, Inc., Ipswich, MA, USA), and the BST. + The DNA polymerases used are those of ArcticZymes Technologies ASA (ArcticZymes Technologies ASA, Tromsø, Norway), the SD polymerase of Bioron GmbH (Bioron GmbH, Römerberg, Germany), and the phi29 DNA polymerase of New England Biolabs, Inc. (New England Biolabs, Inc., Ipswich, MA, USA). The SD polymerase, in particular, is a variant of the DNA polymerase of Bacillus stearothermophilus (Bst) modified by one or more mutations.

[0067] Step (c) is typically performed at a temperature of 30 to 72°C. Preferably, step (c) is performed at a temperature of 30 to 65°C, more preferably at 37 to 65°C, and more preferably at 45 to 65°C. Step (c) is typically performed for 5 minutes to 1 hour. Step (c) can also be performed for longer than 1 hour, for example, for 90 minutes or for 2 hours. Preferably, step (c) is performed for 10 minutes to 1 hour, and more preferably for 20 to 30 minutes. For example, step (c) is performed for 20 minutes at 65°C. R.414809

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[0069] The nucleic acids formed in step (c) can also be called preamplifiers.

[0070] After pre-amplification of the nucleic acids, the reaction mixture is heated to 90 to 100°C (step (d)). Preferably, the reaction mixture is heated to 90 to 98°C, more preferably to 93 to 95°C, for example to 95°C. Step (d) is typically carried out for 1 to 30 min. Preferably, step (d) is carried out for 5 to 30 min, more preferably for 10 to 15 min. For example, step (d) is carried out for 10 min at 95°C. The conditions in step (d) are chosen, among other things, depending on the PCR primers used. The PCR primers must assume an active form under the conditions in step (d) so that they are available for the subsequent amplification of the target nucleic acid sequence. This applies accordingly to the PCR polymerase. In contrast, the pre-amplification polymerase must be inactivated under the conditions in step (d).

[0071] In step (d), heating leads to the inactivation of the pre-amplification polymerase, the activation of the PCR polymerase, and the activation of the PCR primers. Additionally, in step (d), denaturation of the nucleic acids present (nucleic acids of the starting material as well as pre-amplifiers obtained in step (c)) takes place.

[0072] The pre-amplification primers are typically not inactivated by step (d); they usually remain in their active form. This is not a problem for the subsequent amplification of the target nucleic acid sequence by PCR because, due to their lower melting point compared to PCR primers, the pre-amplification primers do not bind to nucleic acids during PCR. Therefore, the pre-amplification primers do not participate in the PCR. Furthermore, the concentration of the pre-amplification primers during PCR is typically lower than the concentration of the PCR primers because some of the pre-amplification primers are consumed during the pre-amplification of the nucleic acids.

[0073] After heating the reaction mixture to 90 to 100°C, the target nucleic acid sequence is amplified by PCR using PCR primers and R.414809.

[0074] 13 of the PCR polymerase (step (e)). PCR (polymerase chain reaction) is a well-known DNA amplification technique with various known variants, resulting in multiple copies of the target nucleic acid sequence (i.e., a specific DNA segment from a DNA template). PCR has several cycles, each cycle consisting of three steps: denaturation, PCR primer binding, and PCR primer elongation. These three steps take place at different temperatures. During PCR primer elongation, a PCR polymerase continuously adds single nucleotides to the PCR primer. The PCR polymerase is a DNA polymerase. Examples of suitable PCR polymerases are AmpliTaq Gold DNA polymerase from Life Technologies GmbH (Life Technologies GmbH, Darmstadt, Germany) and the DNA polymerases described in WO 2009 / 155464 A2 as 505 R and 540D. A standard PCR protocol can be used for PCR.An example of a standard PCR protocol comprises 25 to 40 cycles, each with denaturation at 95°C for 30 s, PCR primer binding at 55-68°C for 1 min, and PCR primer elongation at 72°C for 3 min. The PCR can, for example, be a quantitative PCR.

[0075] Because all the necessary reagents are present in the reaction chamber from the beginning, steps (c) to (e) in the procedure can follow each other immediately.

[0076] By combining pre-amplification and amplification, the method is suitable for small amounts of starting material, such as a single cell. Therefore, the method is particularly suitable for analyzing cells, for example, circulating tumor cells, at the single-cell level.

[0077] By carrying out the process in a single reaction chamber, the process can be easily integrated into a microcavity array.

[0078] Furthermore, the method can amplify both target nucleic acid sequences in the form of RNA and target nucleic acid sequences in the form of DNA. Therefore, the method has a broad range of applications.

[0079] According to another embodiment, no reagents are added or removed during the process. This means, in particular, that between R.414809

[0080] No purification, rebuffering, or similar steps are required in the first and subsequent second reactions. This makes the process particularly easy to automate. Therefore, the method is especially suitable for point-of-care analyses and patient-side diagnostic tests.

[0081] Since no reagents are added or removed during the process, no additional equipment is required for such operations. This saves material and simplifies the procedure, which is particularly advantageous for point-of-care analyses and patient-specific diagnostic tests.

[0082] Furthermore, carrying out the procedure in a single reaction chamber without the intermediate addition or removal of reagents results in no or only an extremely low loss of the limited nucleic acids of the starting material during the chain of process steps. This can be of great advantage for the quality of the work, especially when working with a small amount of starting material, as is the case, for example, when working at the single-cell level.

[0083] According to another embodiment, steps (c) to (e) are carried out immediately consecutively. This means, in particular, that no purification, re-buffering, or the like takes place between steps (c) and (e). This makes the process particularly easy to automate.

[0084] According to another embodiment, the starting material is a single cell. As described above, the method, through the combination of pre-amplification and amplification, is also suitable for a small quantity of starting material, such as a single cell. The single cell, which can also be referred to as a single cell, is in particular a tumor cell. The tumor cell is, in particular, a circulating tumor cell. A circulating tumor cell (CTC) is a tumor cell that has separated from a solid primary tumor and is detectable in the blood. The analysis of circulating tumor cells is of great scientific and medical interest, as the cells can serve, for example, as a predictive marker for the further course of the disease or for the response to a specific cancer therapy. It is also assumed that circulating tumor cells are responsible for the development of tumors. R.414809

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[0086] Metastases are responsible. The method according to the invention is particularly well suited for the analysis of circulating tumor cells at the single-cell level.

[0087] The individual cells can be obtained from a sample, such as a blood sample, prior to the procedure by targeted enrichment. This enrichment can be achieved, for example, using a microsphere, particularly a magnetic microsphere, to which an antibody specific for a particular surface molecule of the desired type of individual cells is coupled. Such methods are known and are also used for the targeted enrichment of circulating tumor cells from a blood sample obtained from a cancer patient. Typically, magnetic microspheres coupled with an antibody specific for a particular surface molecule of the circulating tumor cells are used for the enrichment of circulating tumor cells from a blood sample.In primary tumors of epithelial origin, the surface molecule specific to circulating tumor cells can be, for example, EpCAM. Instead of the antibody, another affinity reagent specific to a particular surface molecule of circulating tumor cells can be used. Alternatively, magnetic beads that bind specifically to blood cells, especially leukocytes, are employed. These beads serve to remove leukocytes from the blood sample and are accordingly modified with an affinity reagent specific to a particular leukocyte surface molecule, such as CD45.

[0088] Methods that do not require microspheres are also possible for enriching the desired cells. For example, circulating tumor cells can be separated from blood cells by filtration or microfluidic geometries.

[0089] Furthermore, the cell used as starting material may have been obtained without prior enrichment or selection. This is the case, for example, when the method is used for single-cell analysis of tumor cells from tumor tissue.

[0090] For single-cell analysis, the cells are isolated before the procedure. Isolation can occur, for example, during the introduction of the cells into the reaction chambers. R.414809

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[0092] As an alternative to a single cell, the starting material can also consist of multiple cells. The process is suitable for both single cells and multiple cells as starting material.

[0093] Before the procedure is carried out, the cells are introduced into the reaction chambers, with only one cell being introduced into each chamber for single-cell analysis. This can be achieved, for example, using a statistical process. The introduction of the cell into the reaction chamber can be accomplished, for instance, by gravity; that is, the cell is allowed to settle into the reaction chamber.

[0094] Before starting the process, further steps can be performed, for example, to remove any cells from the reaction chamber that are not bound to the magnetic bead used for the enrichment of the desired cells. For instance, after the cells have settled into the reaction chamber, a washing step can be carried out to remove unbound beads. A magnetic field can then be applied to the bottom of the reaction chamber to hold the remaining bead with the bound cell in place. The reaction chamber can then be rotated 180°, causing any cells not bound to the magnetic bead to leave the chamber due to gravity. These cells can then be removed by another washing step. After another 180° rotation of the reaction chamber, further preparation can be carried out, if necessary.Finally, the reaction chamber can be sealed. The process can now be started.

[0095] According to another embodiment, the pre-amplification polymerase is bound to the surface of a bead. In other words, the pre-amplification polymerase is immobilized on the surface of a bead. The bead is, in particular, a magnetic bead. The magnetic bead can be the one used for the enrichment of the desired cells. However, the bead can also be a different bead. The pre-amplification polymerase can be active on the surface of the bead. In this case, the linker that connects the immobilized pre-amplification polymerase to the surface is selected such that the pre-amplification polymerase is active on the surface of the bead.

[0096] 17. The pre-amplification polymerase exists in an active form or in an inactive form that can be activated under the conditions in step (b). Alternatively, it is conceivable that the pre-amplification polymerase detaches from the surface of the bead under the conditions in step (b) and thus acquires an active form.

[0097] Immobilizing the pre-amplification polymerase offers another way to prevent cells that are not bound to the magnetic beads used for cell enrichment from being used as starting material for the process. For this purpose, the pre-amplification polymerase can be immobilized on the surface of the magnetic beads used for cell enrichment. This prevents the process from running if even one or more cells that are not bound to the magnetic beads used for cell enrichment are present in the reaction chamber. Unbound beads can be removed from the reaction chamber by a washing step before starting the process.

[0098] According to a further embodiment, the reaction space is at least partially bounded by a solid phase. Such a reaction space can, in particular, be a cavity of a microcavity array. The cavity can, for example, accommodate a reaction volume in the picoliter (pl) range, so that the reaction volume can be, for example, 8 pl or 42 pl. When filling several cavities of an array with their respective starting material, it is important to ensure that reagents already present within the individual cavities are not carried over or transferred, since the cavities are connected to each other via the liquid front during this phase. This can be achieved, for example, by flushing the cavities with oil. Flushing the cavities with oil creates separate reaction spaces. At the same time, the cavities are sealed by flushing with oil.

[0099] According to another embodiment, the pre-amplification polymerase is present in an inactive form in step (a) and can be activated under the conditions in step (b). This prevents non-specific incorporation of dNTPs by the pre-amplification polymerase before step (b). In this case, the pre-amplification polymerase is a temperature-activated pre-amplification polymerase. R.414809

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[0101] According to an alternative embodiment, the pre-amplification polymerase is present in an active form in step (a).

[0102] According to another embodiment, the method further comprises:

[0103] Cooling the reaction mixture after step (b) and before step (c).

[0104] This step can facilitate the binding of the pre-amplification primers to the nucleic acids of the starting material and thus the initiation of the isothermal amplification in step (c). Cooling the reaction mixture can also serve to bring it to the temperature at which the isothermal amplification is carried out. Alternatively, cooling can be performed to a temperature below the temperature at which the isothermal amplification takes place.

[0105] Typically, the reaction mixture is cooled to a temperature of 4 to 50°C to aid the binding of the pre-amplification primers to the nucleic acids of the starting material. Preferably, the reaction mixture is cooled to 10 to 50°C, more preferably to 20 to 45°C. The step of cooling the reaction mixture after step (b) and before step (c) is typically carried out for 10 seconds to 10 minutes. Preferably, the step is carried out for 10 seconds to 1 minute, more preferably for 10 to 30 seconds. For example, the step is carried out for 30 seconds at 30°C.

[0106] According to another or alternative embodiment, the method before step (c) further comprises:

[0107] Heating the reaction mixture.

[0108] Heating the reaction mixture can be used to bring it to the temperature at which isothermal amplification is carried out. R.414809

[0109] 19

[0110] According to another embodiment, isothermal amplification is a strand displacement amplification. Strand displacement amplification is particularly suitable for pre-amplifying nucleic acids. This is partly because SD polymerases have very similar requirements for a suitable buffer as PCR polymerases. Therefore, a suitable buffer for the process, compatible with both the SD polymerase and the PCR polymerase used in the process, is particularly easy to determine.

[0111] According to another embodiment, the pre-amplification primers are primers with a random sequence. In this case, the pre-amplification can be performed as a universal pre-amplification.

[0112] According to an alternative embodiment, the pre-amplification primers include both primers with a random sequence and primers specific to the target nucleic acid sequence. In this case, the pre-amplification can be performed as a semi-specific pre-amplification.

[0113] According to another embodiment, the PCR primers are bound to a surface of the reaction chamber by a temperature-labile covalent bond up to step (d). In other words, the PCR primers are immobilized to a surface of the reaction chamber by a temperature-labile covalent bond up to step (d). As a result, the PCR primers are in an inactive form up to step (d). The reaction chamber is thus surface-functionalized. The term "temperature-labile bond," as used here, means that the bond withstands the conditions in step (b) but not the conditions in step (d) and therefore breaks down in step (d). Accordingly, the temperature-labile covalent bond to the surface of the reaction chamber is broken in step (d). By heating the reaction mixture in step (d), the PCR primers are thus dissolved and thereby converted into an active form.

[0114] To bind PCR primers to a surface via a temperature-labile covalent bond, PCR primers with a temperature-labile chemical modification can serve as a starting point. Temperature-labile chemical modifications of PCR primers are known and are used to increase the specificity of primer binding. For example, it is known to functionalize a phosphate bridge within a PCR primer or to extend it by a side chain, for example, R.414809.

[0115] 20 with a 4-oxo-tetradecyl group, a 4-oxo-pentyl (OXP) group, or a 2-(N-formyl-N-methyl)aminoethyl (MAF) group. Multiple phosphate bridges, for example, two phosphate bridges, within a PCR primer can also be modified in this way. Heating the reaction mixture cleaves off the temperature-labile chemical modification, leaving the PCR primer in its unmodified form.

[0116] To bind PCR primers to a surface via a temperature-labile covalent bond, the described temperature-labile chemical modification can be extended by an additional chemical modification, such as a terminal thiol group or a terminal primary amine. This additional chemical modification is used to bind the PCR primers to the surface, thus achieving a temperature-labile covalent bond. For example, a thiol group can react with maleimide in aqueous solution. If a bismaleimide, such as phenylbismaleimide, is used, the first maleimide group of the bismaleimide can react with the thiol group of the PCR primer in a first Michel addition, and the second maleimide group of the bismaleimide can be used to react with thiol groups present on the surface in a second Michel addition.Finally, the two now thiosubstituted maleimide groups of the bismaleimide undergo hydrolysis. The surface can be, for example, a silicon surface modified (functionalized) with thiol groups. In the case of a primary amine as an additional chemical modification, carboxyl, aldehyde, sulfonic acid, epoxy, and / or isothiocyanate groups, for example, can be used as surface modifications to achieve binding of the PCR primers to the surface. Suitable surfaces include, for example, appropriately functionalized silicon chips or modified polymers. Therefore, at least for the area in which the PCR primers are immobilized prior to the procedure, a suitably suitable or appropriately functionalized surface must be selected for the reaction chamber.

[0117] According to an alternative embodiment, the PCR primers are bound to a surface of a bead by means of a temperature-labile covalent bond up to step (d). In other words, the PCR primers are immobilized to a surface of the bead by means of a temperature-labile covalent bond up to step (d). Thus, the PCR primers are in a state of R.414809 up to step (d).

[0118] 21 inactive form. The temperature-labile covalent bond to the surface of the bead is broken in step (d). By heating the reaction mixture in step (d), the PCR primers are thus dissolved and thereby converted into an active form. The bead is, in particular, a magnetic bead. The magnetic bead can be the one used for the enrichment of the desired cells. However, the bead can also be a different bead. The possibilities described above for the temperature-labile covalent bonding of the PCR primers to a surface apply accordingly to the temperature-labile covalent bonding of the PCR primers to the surface of a bead.

[0119] As previously described, PCR primers can have a barcode or identification sequence to enable subsequent assignment of the amplicons to the individual reaction compartments and thus to the respective starting material. Therefore, an individual barcode sequence is used for each reaction compartment. If such primers are bound to the surface of a bead—used prior to the procedure for enriching the desired cells—by a temperature-labile covalent bond up to step (d), then an individual barcode sequence is used for each bead. In this case, it is essential to prevent multiple beads from binding to a single cell and thus introducing different barcode sequences into the reaction compartment. For unambiguous barcoding, it must be ensured that an individual barcode sequence is incorporated into the amplicons for each individual cell.

[0120] According to another alternative embodiment, the PCR primers are bound to oligonucleotides complementary to the PCR primers by base pairing up to step (d), wherein the oligonucleotides complementary to the PCR primers are bound to a surface of the reaction chamber by a temperature-stable covalent bond. In this embodiment, it is not the PCR primers themselves, but rather the oligonucleotides complementary to the PCR primers that are immobilized to a surface of the reaction chamber. The term "temperature-stable bond" as used here means that the bond withstands both the conditions in step (b) and the conditions in step (d) and thus does not break down in step (d). Such temperature-stable bonds are known and can also be referred to as irreversible bonds. The base pairing between the PCR primers and the oligonucleotides complementary to them maintains the R.414809

[0121] 22

[0122] The conditions in step (b) are fixed and are separated in step (d). This can be achieved, for example, by selecting a sufficiently long segment for base pairing between the PCR primers and their complementary oligonucleotides. As a result, the PCR primers are inactive until step (d). Heating the reaction mixture in step (d) dissolves the PCR primers, thus converting them into an active form.

[0123] The oligonucleotides complementary to the PCR primers can be terminally modified with a thiol group or a primary amine to bind the oligonucleotides to the surface. As described above, a thiol group can react with maleimide in aqueous solution. If a bismaleimide, such as phenyl bismaleimide, is used, the first maleimide group of the bismaleimide can react with the thiol group of the oligonucleotide, and the second maleimide group of the bismaleimide can be used to react with thiol groups present on the surface. In the case of a primary amine, carboxyl, aldehyde, sulfonic acid, epoxy, and / or isothiocyanate groups, for example, can be used as surface modifications to achieve binding of the oligonucleotides to the surface. Suitable surfaces include, for example, functionalized silicon chips or modified polymers.Therefore, at least for an area in which the oligonucleotides are immobilized prior to the process, a suitably appropriate or functionalized surface must be selected as the surface of the reaction space.

[0124] In this embodiment, the oligonucleotides are irreversibly immobilized on the surface of the reaction chamber prior to the process. The PCR primers can then be added to the reaction chamber. After heating to 90–100°C and subsequent cooling, for example to room temperature, the PCR primers bind to the immobilized oligonucleotides via base pairing. Unbound PCR primers can be removed by a washing step, which can be performed, for example, at room temperature. To increase the binding capacity of the reaction chamber surface, the oligonucleotides can exhibit the sequence complementary to the PCR primers multiple times. To prevent unwanted reactions at the resulting double-stranded segments, the sequence repeats can be separated from each other using non-natural nucleic bases. For example, the sequence repeats R.414809

[0125] 23 are separated from each other using PEG (polyethylene glycol) spacers. In this way, the double-stranded sections are not accepted by a polymerase as a DNA template.

[0126] The oligonucleotides can be structured as peptide nucleic acids (PNAs). This also prevents unwanted reactions at the oligonucleotides, especially at the resulting double-stranded segments.

[0127] According to another alternative embodiment, the PCR primers are in an inactive form up to step (d) due to a temperature-labile chemical modification. Such PCR primers are known. In this embodiment, the PCR primers can be in solution from the beginning of the process. Therefore, this embodiment is also suitable for use in droplets as the reaction chamber. The term "temperature-labile chemical modification," as used here, means that the chemical modification withstands the conditions in step (b) but not the conditions in step (d) and thus detaches, for example, in step (d). Accordingly, the temperature-labile chemical modification is removed in step (d). By heating the reaction mixture in step (d), the PCR primers are thus converted into an active form.An example of such PCR primers are the so-called CleanAmp primers from TriLink BioTechnologies (TriLink BioTechnologies, San Diego, CA, USA).

[0128] According to another embodiment, the reagents comprise a nickase, and the pre-amplification primers have a recognition sequence for the nickase. In this case, isothermal amplification can take place using the nickase. A nickase (also called a nicking enzyme) is an endonuclease that creates a single-strand break at a specific position within a double-stranded DNA molecule. The recognition sequence is specific to a particular nickase.

[0129] The use of a nickase for isothermal amplification, particularly isothermal SDA, is known. Once a double-stranded recognition sequence for the nickase has been generated during SDA, the nickase creates a single-strand break. At the site of the single-strand break, DNA polymerase R.414809 can be used again.

[0130] 24 bind and synthesize the complementary strand, while the already existing complementary strand is displaced.

[0131] A nickase is selected that (i) exists in step (a) in an active form or in an inactive form that can be activated under the conditions in step (b), (ii) remains stable under the conditions in step (b), (iii) is active at the temperature at which the isothermal amplification is carried out, and (iv) can be inactivated under the conditions in step (d). In this way, the nickase is available for the pre-amplification of the nucleic acids. During the process, the nickase is inactivated by appropriate heating of the reaction mixture. This prevents the nickase from being available for the second reaction. Examples of suitable nickases are Nt.BbvCI, Nt.BspQI, Nt.BstNBI, Nb.BsrDI, Nb.BtsI, Nt.Alwl, Nb.BbvCI, and Nb.Bsml, all from New England Biolabs, Inc. (New England Biolabs, Inc., Ipswich, MA, USA).

[0132] According to another embodiment, the nucleic acids of the starting material comprise DNA and RNA.

[0133] According to another embodiment, the reagents comprise a reverse transcriptase, wherein the reverse transcriptase remains stable under the conditions in step (b), and wherein step (c) comprises the transcription of nucleic acids present in the form of RNA into complementary DNA by reverse transcription using the reverse transcriptase. In this case, isothermal amplification can thus take place together with the transcription of nucleic acids present in the form of RNA into complementary DNA (cDNA) by reverse transcription. This is particularly necessary when the target nucleic acid sequence is present in the form of RNA, for example, in the form of mRNA, and the chosen pre-amplification polymerase can only amplify DNA. Reverse transcription is a well-known molecular biology technique.

[0134] A reverse transcriptase is chosen that is active at the temperature at which isothermal amplification is performed. The reverse transcriptase can be in an active form in step (a) or in an inactive form that can be activated under the conditions in step (b). In this way, the reverse transcriptase is available for the pre-amplification of the nucleic acids. Examples for R.414809

[0135] 25 suitable reverse transcriptases are the RapiDxFire Thermostable Reverse Transcriptase from BioCat GmbH (BioCat GmbH, Heidelberg, Germany) and the Re-vTaq RT-PCR DNA Polymerase from myPOLS Biotec GmbH (myPOLS Biotec GmbH, Konstanz, Germany).

[0136] According to an alternative embodiment, a pre-amplification polymerase is chosen that can amplify both RNA and DNA. An example of such a pre-amplification polymerase is the Bst 3.0 DNA polymerase from New England Biolabs, Inc. (New England Biolabs, Inc., Ipswich, MA, USA), which also exhibits reverse transcriptase activity.

[0137] According to an alternative embodiment, a pre-amplification polymerase is chosen that can amplify both RNA and DNA, and the reagents additionally include a reverse transcriptase, the reverse transcriptase remaining stable under the conditions in step (b). In this way, the pre-amplification of RNA is optimally supported.

[0138] According to a further embodiment, step (e) additionally includes the detection of the amplicons of the target nucleic acid sequence obtained in this step. This enables real-time detection of the amplicons of the target nucleic acid sequence. The amplicons can, for example, be detected optically in real time. Step (e) can be performed, for example, using a detection probe that is specific for the target nucleic acid sequence. According to a further embodiment, the reagents therefore comprise a detection probe for detecting the amplicons of the target nucleic acid sequence obtained in step (e).A detection probe, which may also be called a hybridization probe or detection probe, is an oligonucleotide complementary to a segment of a target nucleic acid sequence to be amplified and which carries a tag that, upon binding of the oligonucleotide to this segment, generates a detectable signal, in particular an optically detectable signal. The optically detectable signal may, for example, be a fluorescence signal. The detection probe is chosen such that it is not active in step (b) and is therefore not consumed in step (b). The detection probe may, for example, be a molecular beacon. R.414809.

[0139] 26

[0140] According to another embodiment, a detection probe is selected that (i) is in an inactive form in step (a), (ii) remains inactive under the conditions in step (b), and (iii) can be activated under the conditions in step (d). In this way, the detection probe is only available in step (e). Up to step (d), the detection probe can be bound, for example, by a temperature-labile covalent bond to a surface of the reaction chamber or to the surface of a bead. The temperature-labile covalent bond is broken in step (d). By heating the reaction mixture in step (d), the detection probe is thus dissolved and thereby activated. A temperature-labile covalent bond to a surface of the reaction chamber or to the surface of a bead has already been described in more detail for PCR primers.The detection probe can be bound to the surface in the same way as the PCR primers. Furthermore, the PCR primers and the detection probe can be bound to the same surface.

[0141] Alternatively, the amplicons of the target nucleic acid sequence obtained in step (e) can be detected by a downstream detection method. Such detection methods are known. These include, for example, detection of the amplicons by DNA gel electrophoresis (agarose gel electrophoresis) or detection of the amplicons by hybridization on microarrays.

[0142] Depending on the desired further use of the target nucleic acid sequence amplicons obtained in step (e), the reaction mixture can be cooled after the procedure. This allows the amplicons to be used or stored for a downstream detection method or for other purposes. Typically, the reaction mixture is cooled to 4°C.

[0143] Depending on the desired further use of the target nucleic acid sequence amplicons obtained in step (e), the amplicons can be purified by a downstream purification procedure, which can also be referred to as a selection procedure. Suitable purification procedures are known. Purification removes, for example, the nucleic acids that were available as DNA templates in step (e) from the target nucleic acid sequence amplicons obtained in step (e). Any remaining pre-amplification primers are also removed from the target nucleic acid sequence amplicons obtained in step (e) by purification. Furthermore, R.414809

[0144] 27 The purification procedure is adjusted so that even non-elongated PCR primers, due to their comparatively short length, are removed from the target nucleic acid sequence amplicons obtained in step (e). Purification allows subsequent steps for analyzing the target nucleic acid sequence, such as sequencing, to be performed more efficiently. The purification procedure may involve combining the amplicons from several reaction compartments.

[0145] The purification process can be carried out, for example, using magnetic beads. Suitable magnetic beads for this purpose are known. These include, for example, the AMPure XP-Beads from Beckman Coulter Life Sciences (Beckman Coulter Life Sciences, Indianapolis, IN, USA).

[0146] If the reaction chamber is sealed by an oil layer, the oil phase can be displaced by an aqueous, buffered phase at the beginning of the purification process. This aqueous, buffered phase may contain purification reagents such as magnetic beads suitable for purification.

[0147] According to another embodiment, the PCR primers are biotinylated. The biotinylated PCR primers cause the amplicons of the target nucleic acid sequence obtained in step (e) to contain a biotin molecule and can therefore be purified by streptavidin purification. The streptavidin purification can be carried out, for example, using streptavidin-coupled beads, in particular streptavidin-coupled magnetic beads. The interaction between streptavidin and biotin enables the purification of the amplicons obtained in step (e).

[0148] The amplicons of the target nucleic acid sequence, purified by the downstream purification process, can, for example, be subjected to sequencing.

[0149] According to another embodiment, the reagents comprise a plurality of pairs of PCR primers, each in an inactive form, remaining inactive under the conditions in step (b) and under the conditions in R.414809

[0150] 28

[0151] Step (d) can be activated. The pairs of PCR primers can be specific for different target nucleic acid sequences. The previously disclosed explanations regarding the PCR primers apply accordingly when using multiple pairs of PCR primers. By using multiple pairs of PCR primers, multiple target nucleic acid sequences can be amplified simultaneously in step (e) (multiplex PCR).

[0152] The invention further relates to a kit for amplifying a target nucleic acid sequence from a starting material comprising nucleic acids, the kit comprising:

[0153] (i) a plurality of pre-amplification primers,

[0154] (ii) a pre-amplification polymerase,

[0155] (iii) at least one pair of PCR primers, and

[0156] (iv) a PCR polymerase, wherein the pre-amplification primers are in an active form, wherein the pre-amplification polymerase remains stable upon heating to 50 to 85°C and can be inactivated by heating to 90 to 100°C, wherein the PCR primers and the PCR polymerase are each in an inactive form, wherein the PCR primers and the PCR polymerase remain inactive upon heating to 50 to 85°C and can be activated by heating to 90 to 100°C.

[0157] According to another embodiment, the kit further includes:

[0158] (v) a buffer compatible with the pre-amplification polymerase and the PCR polymerase, and

[0159] (vi) Deoxynucleotide triphosphates.

[0160] The further explanations and embodiments disclosed in the description of the method, in particular with regard to the individual components of the kit, apply accordingly to the kit.

[0161] The invention further relates to a use of the kit according to the invention for amplifying a target nucleic acid sequence from a starting material containing nucleic acids.

[0162] The further explanations and embodiments disclosed in the description of the method apply accordingly to the use of the kit. R.414809

[0163] 29

[0164] A method for amplifying a target nucleic acid sequence from a starting material containing nucleic acids has been disclosed, the method comprising:

[0165] (a) Providing a reaction chamber in which the starting material and all reagents for the subsequent process steps (b) to (d) are present, wherein the reagents comprise a plurality of pre-amplification primers, wherein the reagents comprise at least one pair of PCR primers, wherein the reagents comprise a polymerase suitable for pre-amplifying the nucleic acids by isothermal amplification and for amplifying the target nucleic acid sequence by PCR, wherein the starting material together with the reagents forms a reaction mixture,

[0166] (b) Heating the reaction mixture to 50 to 85°C,

[0167] (c) Pre-amplification of the nucleic acids by isothermal amplification using the pre-amplification primers and the polymerase,

[0168] (d) Heating the reaction mixture to 90 to 100°C,

[0169] (e) Amplifying the target nucleic acid sequence by PCR using the PCR primers and the polymerase, wherein the pre-amplification primers are in an active form in step (a), wherein the PCR primers are in an inactive form in step (a), wherein the PCR primers remain inactive under the conditions in step (b) and are activatable under the conditions in step (d).

[0170] The disclosed process uses only a single polymerase. This polymerase must be stable under the conditions in both step (b) and step (d). An example of a polymerase suitable for pre-amplifying nucleic acids by isothermal amplification and amplifying the target nucleic acid sequence by PCR is the SD polymerase from Bioron GmbH (Bioron GmbH, Römerberg, Germany). This polymerase is stable up to 93°C, so when using this polymerase in step (d), heating is only required to 90–92°C. R.414809

[0171] 30

[0172] The further explanations and embodiments disclosed in the description of the method according to the invention apply accordingly to the disclosed method.

[0173] The invention will now be explained using the figures. They show:

[0174] Figure 1 schematically represents a process flow of a method for amplifying a target nucleic acid sequence from a starting material containing nucleic acids.

[0175] Figure 2 shows a schematic representation of a first embodiment of a method according to Figure 1 with a downstream purification step.

[0176] Figure 3 schematically shows a second embodiment of a method according to Figure 1 with a downstream purification step.

[0177] Figure 4 shows a schematic representation of an embodiment of a PCR primer that has a temperature-labile chemical modification.

[0178] Figure 5 shows the chemical structure of the temperature-labile chemical modification of the PCR primer according to Figure 4.

[0179] Figure 6 shows a reaction scheme for the binding of the PCR primer 10 according to Figure 4 to a surface of a reaction chamber.

[0180] Figure 7 shows a reaction scheme for the removal of the PCR primer 10 from the surface of the reaction chamber by heating the reaction mixture.

[0181] Figure β schematically shows further embodiments for binding the PCR primer 10 according to Figure 4 to a surface of a reaction chamber, wherein the surface has several functionalizations that are used in different embodiments. R.414809

[0182] 31

[0183] With reference to Figure 1, a process flow of a method for amplifying a target nucleic acid sequence from a starting material containing nucleic acids is explained.

[0184] In a first step, S200, a reaction chamber is provided containing the starting material and all reagents for the subsequent process steps S300 to S700. In the present embodiment, step S200 is carried out at a temperature of 20°C. In this embodiment, the reaction chamber is a cavity of a microcavity array. The cavity is sealed by a layer of oil and remains sealed for the entire duration of the process.

[0185] The reagents comprise a plurality of pre-amplification primers, a pre-amplification polymerase, a pair of PCR primers, and a PCR polymerase. The reagents further include a buffer compatible with the pre-amplification polymerase and the PCR polymerase, as well as nucleotides (deoxynucleotide triphosphates (dNTPs)) of the types deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), and deoxythymidine triphosphate (dTTP). The pair of PCR primers consists of a forward primer and a reverse primer. The PCR primers are specific for the target nucleic acid sequence.

[0186] The pre-amplification primers are present in an active form in step S200. The pre-amplification primers are in solution. In this embodiment, the pre-amplification primers are exclusively randomized primers. The concentration of the pre-amplification primers in this embodiment is in the range of 0.1 to 20 pM.

[0187] In this embodiment, the pre-amplification polymerase is also present in an active form in solution in step S200. The pre-amplification polymerase used in this embodiment is the Bst 3.0 DNA polymerase from New England Biolabs, Inc. (New England Biolabs, Inc., Ipswich, MA, USA), which can amplify both RNA and DNA.

[0188] The PCR primers and the PCR polymerase are each present in an inactive form in step S200. The inactive form of the PCR primers is shown in the present execution R.414809.

[0189] 32. This is achieved by immobilizing the PCR primers on a surface. After detaching the PCR primers from the surface in a subsequent step S600, the concentration of the PCR primers in this embodiment is in the range of 0.1 to 20 pM.

[0190] In this embodiment, the inactive form of the PCR polymerase is achieved through a chemical modification of the PCR polymerase. The PCR polymerase used in this embodiment is AmpliTaq Gold DNA polymerase from Life Technologies GmbH (Life Technologies GmbH, Darmstadt, Germany).

[0191] In the present embodiment, the starting material is a single cell, specifically a single circulating tumor cell. This cell is a living cell obtained prior to the process from a blood sample taken from a breast cancer patient. The cell forms a reaction mixture together with the reagents.

[0192] In a further step, S300, the reaction mixture is heated to 80°C for 1 minute. This causes thermal lysis of the cell, releasing the cell's nucleic acids, which include both DNA and RNA. Step S300 also results in a partial breakdown of base pairing in the cell's nucleic acids. This partial breakdown of base pairing promotes the binding of the pre-amplification primers to complementary regions of the cell's nucleic acids.

[0193] In contrast to the present embodiment, the pre-amplification polymerase can be in an inactive form in step S200 and be activated in step S300.

[0194] In a further step, S400, the reaction mixture is cooled to 45°C for 30 s. This causes the pre-amplification primers to bind to complementary sections of the cell's nucleic acids.

[0195] Contrary to the present embodiment, step S400 can also be omitted, for example if step S300 is performed at 50°C R.414809

[0196] 33 and the pre-amplification primers can bind to the nucleic acids of the cell at 50°C.

[0197] In a further step, S500, the nucleic acids are pre-amplified by isothermal amplification using pre-amplification primers and pre-amplification polymerase. In step S500, the pre-amplification primers and pre-amplification polymerase are active, while the PCR primers and PCR polymerase remain inactive. Step S500 takes place for 20 minutes at a constant temperature of 65°C. In this embodiment, the isothermal amplification is a strand displacement amplification. This produces pre-amplifiers of the cell's nucleic acids.

[0198] In a further step, S600, the reaction mixture is heated to 95°C for 10 minutes. This inactivates the pre-amplification polymerase, activates the PCR polymerase, and activates the PCR primers. It also denatures the preamplifiers obtained in step S500.

[0199] In a further step, S700, the target nucleic acid sequence is amplified by PCR (polymerase chain reaction) using PCR primers and PCR polymerase. In step S700, the PCR primers and PCR polymerase are active, while the pre-amplification polymerase is inactive. A standard PCR protocol is used. The PCR comprises 30 cycles, each consisting of (i) denaturation at 95°C for 30 s, (ii) PCR primer binding at 68°C for 1 min, and (iii) PCR primer elongation at 72°C for 3 min. Amplicons of the target nucleic acid sequence are generated in step S700.

[0200] In the present embodiment, no reagents are added or removed during the process.

[0201] Performing the process in a single reaction chamber without the intermediate addition or removal of reagents prevents the loss of the limited nucleic acids in the starting material during the chain of process steps. This ensures high process quality, especially when using a single cell as the starting material. R.414809

[0202] 34

[0203] With reference to Figure 2, a first embodiment of the method for amplifying a target nucleic acid sequence from a starting material containing nucleic acids is now described. The method corresponds to the method according to Figure 1, with the embodiment additionally comprising a purification step downstream of the method.

[0204] Figure 2 shows two cavities 20, 120 of a microcavity array. All reagents for the subsequent process steps S300 to S700 are present in both cavities 20, 120. The cavities 20, 120 are sealed by a layer of oil (not shown).

[0205] In the left-hand cavity 20, in addition to the reagents, there is a single cell 1, which serves as the starting material for the process. The left-hand cavity 20 is thus a reaction chamber, as provided in step S200 of the process. In contrast, the right-hand cavity 120 contains no starting material. Cavity 120 is shown for comparison purposes only.

[0206] The reagents comprise a plurality of pre-amplification primers 50, a pre-amplification polymerase 60, a pair of PCR primers 10, and a PCR polymerase 70. The reagents further include a buffer compatible with the pre-amplification polymerase 60 and the PCR polymerase 70 (not shown), as well as nucleotides (not shown). The pair of PCR primers 10 consists of a forward primer and a reverse primer. The PCR primers 10 are specific for the target nucleic acid sequence.

[0207] The pre-amplification primers 50 are in an active form. The pre-amplification primers 50 are in solution. In the present embodiment, the pre-amplification primers 50 are exclusively randomized primers. The pre-amplification polymerase 60 is also in an active form in solution. The pre-amplification polymerase 60 used in the present embodiment can amplify both RNA and DNA.

[0208] In contrast, PCR primers 10 and PCR polymerase 70 are each present in an inactive form. The inactive form is illustrated by a cross. In the present first embodiment, PCR primers 10 are activated by means of a tem- R.414809

[0209] 35 temperature-labile covalent bonds to a surface of cavities 20, 120. The PCR primers 10 are therefore in an inactive form until step S600. The temperature-labile covalent bonding of the PCR primers 10 to the surface of a cavity will be explained in more detail with reference to Figures 4 to 8.

[0210] Cell 1 is a circulating tumor cell obtained prior to the procedure from a blood sample taken from a breast cancer patient. For this purpose, cell 1 was enriched from the blood sample using an enrichment bead 2. The enrichment bead 2 is a magnetic bead to which an antibody specific for a particular surface molecule of the circulating tumor cell (i.e., the circulating breast cancer cell) is attached. Cell 1 is thus bound to the antibody on the enrichment bead 2.

[0211] The process takes place in a single reaction chamber, which remains sealed for the entire duration of the process. Therefore, the enrichment bead 2 also remains in cavity 20 during the process. It is only omitted from the subsequent process steps for the sake of clarity.

[0212] Cell 1 forms a reaction mixture together with the reagents.

[0213] In step S300, the reaction mixture is heated to 80°C for 1 minute. This causes thermal lysis of cell 1, releasing the nucleic acids of cell 1, which include both DNA 3 and RNA 4. Step S300 also results in a partial breakdown of base pairing in the nucleic acids. This partial breakdown of base pairing promotes the binding of the pre-amplification primers 50 to complementary regions of the nucleic acids. The PCR primers 10 and the PCR polymerase 70 remain in their inactive form during step S300.

[0214] In a further step S400, the reaction mixture is cooled to 45°C for 30 s. This causes the pre-amplification primers 50 to bind to complementary regions of DNA 3 and RNA 4.

[0215] In a further step S500, the DNA 3 and RNA 4 are amplified by isothermal amplification using the pre-amplification primers 50 and R.414809.

[0216] 36 cation polymerases 60 pre-amplify the RNA 4, wherein the pre-amplification of RNA 4 comprises the transcription of RNA 4 into cDNA by reverse transcription. Step S500 takes place for 20 min at a constant temperature of 65°C. In the present embodiment, the isothermal amplification is a strand displacement amplification. This produces pre-amplifiers 5 of the nucleic acids. The PCR primers 10 and the PCR polymerase 70 are still in their inactive form.

[0217] In a further step, S600, the reaction mixture is heated to 95°C for 10 minutes. This inactivates the pre-amplification polymerase 60. The inactive form is illustrated by a cross. In contrast, PCR polymerase 70 and PCR primers 10 are activated by the heating in step S600. The PCR primers 10 are released from the surface of cavities 20 and 120. The PCR primers 10 are thus dissolved and converted into an active form. Furthermore, the pre-amplifiers 5 obtained in step S500 are denatured by the heating in step S600.

[0218] In a further step, the target nucleic acid sequence is amplified by PCR using PCR primers 10 and PCR polymerase 70. Figure 2 shows this step divided into substeps S700a and S700b, where in step S700a the PCR primers 10 bind to complementary regions of the preamplifiers 5. Step S700a thus corresponds to PCR primer binding in the first PCR cycle. Step S700b comprises the PCR primer elongation of the first PCR cycle as well as the subsequent 29 PCR cycles, each consisting of (i) denaturation at 95°C for 30 s, (ii) PCR primer binding at 68°C for 1 min, and (iii) PCR primer elongation at 72°C for 3 min. This results in amplicons 6 of the target nucleic acid sequence.

[0219] In a subsequent purification step 800, the amplicons 6 of the target nucleic acid sequence are separated from the remaining reaction mixture by means of a magnetic purification bead 7. After purification 800, the amplicons 6 can be used, for example, for sequencing the target nucleic acid sequence. R.414809

[0220] 37

[0221] With reference to Figure 3, a second embodiment of the method for amplifying a target nucleic acid sequence from a starting material containing nucleic acids will now be explained. The second embodiment is largely identical to the first embodiment, so the differences between the first and second embodiments will be explained in more detail below.

[0222] Figure 3 shows two cavities 220 and 320 of a microcavity array. Both cavities 220 and 320 contain reagents for the subsequent process steps S300 to S700. The cavities 220 and 320 are sealed by a layer of oil (not shown).

[0223] In the left-hand cavity 220, in addition to the reagents, there is a single cell 1, which serves as the starting material for the process. As in the first embodiment, cell 1 is a circulating tumor cell obtained prior to the process from a blood sample taken from a breast cancer patient. For this purpose, cell 1 was enriched from the blood sample using an enrichment bead 102. The enrichment bead 102 is a magnetic bead to which an antibody specific for a particular surface molecule of the circulating tumor cell (i.e., the circulating breast cancer cell) is coupled. Cell 1 is thus bound to the antibody on the enrichment bead 102.

[0224] In contrast to the first embodiment, in the present second embodiment the PCR primers 110 are bound to a surface of the enrichment bead 102 by means of a temperature-labile covalent bond. The PCR primers 110 are therefore in an inactive form until step S600. The other reagents are identical to those in the first embodiment.

[0225] Due to their binding to the surface of the enrichment bead 102, the PCR primers 110 are present only in the left-hand cavity 220. The left-hand cavity 220 is therefore a reaction chamber, as provided in step S200 of the procedure. In contrast, the right-hand cavity 320 contains neither starting material nor PCR primers 110. Cavity 320 is shown for comparison purposes only. R.414809

[0226] 38

[0227] Cell 1 forms a reaction mixture together with the reagents.

[0228] Steps S300, S400 and S500 are identical to the first embodiment.

[0229] Step S600 is essentially identical to the first embodiment. In contrast to the first embodiment, in this second embodiment the PCR primers 110 are released from the surface of the enrichment bead 102. The PCR primers 110 are thus dissolved and therefore converted into an active form.

[0230] Since the reaction chamber remains sealed for the entire duration of the process, the enrichment bead 102 remains in cavity 220 for the remainder of the process. It is only omitted from step S600 onwards for the sake of clarity.

[0231] Steps S700a and S700b are identical to the first embodiment.

[0232] The downstream purification step 800 also corresponds to the first embodiment.

[0233] Figure 4 schematically depicts an embodiment of a PCR primer 10 that incorporates a temperature-labile chemical modification. The 3' end of the PCR primer 10, containing the nucleic bases Bi to Bs, is shown in detail on the right side of Figure 4. A phosphate bridge within the PCR primer 10 exhibits a functionalization X, which is the temperature-labile chemical modification. This temperature-labile chemical modification withstands the conditions in step S300 of the process but not the conditions in step S600. In contrast to the present embodiment, two or more phosphate bridges within the PCR primer 10 can also be modified in this way.

[0234] Figure 5 shows the chemical structure of the temperature-labile chemical modification (functionalization X) of PCR primer 10 according to Figure 4. It is a substituted 4-oxo-tetradecyl group. This group is attached at its 1-position, located on the right side of Figure 5, to the phosphate bridge of R.414809.

[0235] 39

[0236] PCR primer 10 is attached. Substituent R is located at position 14, which is on the left side of Figure 5. Substituent R is an additional chemical modification, such as a terminal thiol group or a terminal primary amine. This additional chemical modification is used to bind PCR primer 10 to a surface, such as the surface of a reaction chamber.

[0237] Figure 6 shows a reaction scheme for the temperature-labile covalent bonding of the PCR primer 10 according to Figure 4 to the surface of a reaction chamber. In the present embodiment, the reaction chamber is a cavity 20 of a microcavity array. In this embodiment, the substituent R of the PCR primer 10 is a terminal thiol group. The PCR primer 10 is combined with phenylbismaleimide to form bismaleimide 30. In a first Michel addition 41, the thiol group of the PCR primer 10 is added to the carbon-carbon double bond of a maleimide group of the bismaleimide 30. The PCR primer 10, thus further functionalized, is then filled into the cavity 20, which in this embodiment is part of a silicon microcavity array. The surface of the cavity 20 is functionalized with terminal thiol groups 21.In a second Michel addition 42, the previously unreacted carbon-carbon double bond of the second maleimide group of bismaleimide 30 reacts with one of these thiol groups 21. Finally, hydrolysis 43 of the two now thiosubstituted maleimide groups of bismaleimide 30 takes place. This yields a PCR primer 10, which is bound (immobilized) to the surface of the reaction chamber by a temperature-labile covalent bond until step S600 of the procedure. Thus, the PCR primer 10 exists in an inactive form until step S600.

[0238] Figure 7 illustrates how, in step S600 of the process, heating the reaction mixture cleaves off the temperature-labile chemical modification of the PCR primer 10 (cleavage 44), thereby releasing the PCR primer 10 from the surface of the cavity 20. Step S600 thus dissolves the PCR primer 10 and converts it into an active form. The PCR primer 10 is now in its unmodified form.

[0239] Figure 8 schematically shows several further embodiments of a temperature-labile covalent bonding of the PCR primer 10 according to Figure 4 to an R.414809

[0240] 40

[0241] The surface of a cavity 20 is depicted as a reaction chamber. In these embodiments, the substituent R of the PCR primer 10 is a terminal primary amine, and the surface of the cavity 20 is functionalized with reactive groups 22 to 26. These groups can be a carboxyl group 22, an aldehyde group 23, a sulfonic acid group 24, an epoxy group 25, or an isothiocyanate group 26. A reaction of the primary amine of the PCR primer 10 with one of the reactive groups 22 to 26 yields a PCR primer 10 that is bound (immobilized) to the surface of the reaction chamber by a temperature-labile covalent bond until step S600. Thus, the PCR primer 10 exists in an inactive form until step S600. By heating the reaction mixture in step S600, the temperature-labile chemical modification of the PCR primer 10 is cleaved, thereby releasing the PCR primer 10 from the surface of the cavity 20.Step S600 dissolves PCR primer 10, thus converting it into an active form. PCR primer 10 is now in its unmodified form.

[0242] In contrast to the present embodiments, a 2-(N-formyl-N-methyl)aminoethyl group can function as a thermolabile chemical modification of the PCR primer 10, wherein its formyl group, acting as an aldehyde functionality, facilitates the binding of the PCR primer 10 to the cavity 20. Alternatively, other thermolabile chemical modifications of the PCR primer 10 are possible.

[0243] In contrast to the embodiments shown in Figures 6 to 8, a temperature-labile covalent bond of the PCR primer 10 can be attached to the surface of a bead according to Figure 4. The embodiments described for the surface of a reaction chamber are also suitable for a correspondingly functionalized surface of a bead. This allows a PCR primer 10 to be provided that is bound (immobilized) to the surface of the bead by means of a temperature-labile covalent bond up to step S600. Thus, the PCR primer 10 is in an inactive form up to step S600. The bead can, in particular, be a magnetic bead, for example, a magnetic enrichment bead used for the enrichment of the desired cells, as shown in Figure 3.By heating the reaction mixture in step S600, the temperature-labile chemical modification of PCR primer 10 is cleaved, thereby releasing PCR primer 10 from the surface of the bead. Thus, in step S600, PCR primer 10 is converted into R.414809.

[0244] 41

[0245] The solution is dissolved and thus converted into an active form. PCR primer 10 is now in its unmodified form.

[0246] In another embodiment, it is not the PCR primer itself, but rather an oligonucleotide complementary to the PCR primer that is covalently bound to a surface of the reaction chamber, and this bond is irreversible upon heating. The oligonucleotide is thus bound to the surface by a temperature-stable covalent bond. For example, the oligonucleotide can be terminally modified with a thiol group or a primary amine to bind to the surface. The chemical processes are the same as those described in Figures 6 and 8 for the binding of a PCR primer with a temperature-labile chemical modification to a surface of the reaction chamber, except that the oligonucleotide in this case does not have a temperature-labile chemical modification. The oligonucleotide forms a temperature-stable covalent bond with the surface.After the oligonucleotide binds to the surface of the reaction chamber, the PCR primer is introduced. Heating to 95°C and then cooling to below 50°C induces base pairing between the PCR primer and the oligonucleotide. To prevent unwanted reactions at the resulting double-stranded segments, the oligonucleotide can be a peptide nucleic acid or contain non-natural nucleic bases. Up to step S600, the PCR primer is bound to the oligonucleotide via base pairing and is therefore inactive. Heating the reaction mixture in step S600 breaks the base pairing, releasing the PCR primer. Step S600 thus dissolves the PCR primer into its active form.

[0247] In another embodiment, the PCR primer 10 shown in Figure 4 has an unsubstituted 4-oxo-tetradecyl group as a temperature-labile chemical modification and exists in this inactive form in solution. Heating the reaction mixture in step S600 cleaves off the temperature-labile chemical modification, thus converting the PCR primer 10 into an active, unmodified form. R.414809

[0248] 42

[0249] All described procedures can be used for both PCR primers of the PCR primer pair, that is, for both the forward and reverse primers. Typically, the forward and reverse primers are inactivated in the same way.

Claims

R.414809 43 Patent claims 1. Method for amplifying a target nucleic acid sequence from a starting material containing nucleic acids, the method comprising: (a) Providing a reaction chamber in which the starting material and all reagents for the subsequent process steps (b) to (e) are present, wherein the reagents comprise a plurality of pre-amplification primers (50) and a pre-amplification polymerase (60), wherein the reagents comprise at least one pair of PCR primers (10, 110) and a PCR polymerase (70), wherein the starting material together with the reagents forms a reaction mixture, (b) Heating the reaction mixture to 50 to 85°C, (c) Pre-amplification of nucleic acids by isothermal amplification using the pre-amplification primer (50) and the pre-amplification polymerase (60), (d) Heating the reaction mixture to 90 to 100°C, (e) Amplifying the target nucleic acid sequence by PCR using the PCR primers (10, 110) and the PCR polymerase (70), wherein the pre-amplification primers (50) are in an active form in step (a), wherein the pre-amplification polymerase (60) remains stable under the conditions in step (b) and is inactivated under the conditions in step (d), wherein the PCR primers (10, 110) and the PCR polymerase (70) in the step (a) each in an inactive form, wherein the PCR primers (10, 110) and the PCR polymerase (70) are each under the conditions in the step (b) remain inactive and can be activated under the conditions in step (d).

2. The method according to claim 1, wherein no addition or removal of reagents takes place during the method. R.414809 44 3. Method according to claim 1 or 2, wherein the starting material is a single cell (1).

4. A method according to any one of claims 1 to 3, wherein the pre-amplification polymerase (60) is in an inactive form in step (a) and is activatable under the conditions in step (b).

5. Method according to any one of claims 1 to 3, wherein the pre-amplification polymerase (60) is in an active form in step (a).

6. Method according to any one of claims 1 to 5, further comprising: Cooling the reaction mixture after step (b) and before step (c).

7. Method according to any one of claims 1 to 6, wherein the isothermal amplification is a strand displacement amplification.

8. Method according to any one of claims 1 to 7, wherein the PCR primers (10, 110) are bound to a surface of the reaction chamber by means of a temperature-labile covalent bond up to step (d).

9. Method according to any one of claims 1 to 7, wherein the PCR primers (10, 110) are bound to a surface of a bead up to step (d) by means of a temperature-labile covalent bond.

10. Method according to any one of claims 1 to 7, wherein the PCR primers (10, 110) are bound to oligonucleotides complementary to the PCR primers (10, 110) by base pairing up to step (d), wherein the oligonucleotides complementary to the PCR primers (10, 110) are bound to a surface of the reaction chamber by means of a temperature-stable covalent bond.

11. Method according to any one of claims 1 to 7, wherein the PCR primers (10, 110) are in an inactive form up to step (d) due to a temperature-labile chemical modification. R.414809 45 12. Method according to any one of claims 1 to 11, wherein the pre-amplification primers (50) have a lower melting temperature compared to the PCR primers (10, 110).

13. A method according to any one of claims 1 to 12, wherein the reagents comprise a reverse transcriptase, wherein the reverse transcriptase remains stable under the conditions in step (b), and wherein step (c) comprises rewriting nucleic acids present in the form of RNA into complementary DNA by means of reverse transcription using the reverse transcriptase.

14. Kit for amplifying a target nucleic acid sequence from a starting material containing nucleic acids, the kit comprising: (i) a plurality of pre-amplification primers (50), (ii) a pre-amplification polymerase (60), (iii) at least one pair of PCR primers (10, 110), and (iv) a PCR polymerase (70), wherein the pre-amplification primers (50) are in an active form, wherein the pre-amplification polymerase (60) remains stable upon heating to 50 to 85°C and can be inactivated by heating to 90 to 100°C, wherein the PCR primers (10, 110) and the PCR polymerase (70) are each in an inactive form, wherein the PCR primers (10, 110) and the PCR polymerase (70) each remain inactive upon heating to 50 to 85°C and can be activated by heating to 90 to 100°C.

15. Use of the kit according to claim 14 for amplifying a target nucleic acid sequence from a starting material comprising nucleic acids.

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