Heat-stable DNA polymerases and reverse transcriptases

Abstract

The present invention relates to mutated DNA polymerases derived from wild-type Thermus aquaticus (Taq) DNA polymerase, methods for reverse transcription polymerase chain reaction (RT-PCR) using said DNA polymerases, and kits comprising said DNA polymerases.

Description

[0001] Heat-stable DNA polymerases and reverse transcriptases

[0002] The present invention relates to mutated DNA polymerases derived from wild-type Thermus aquaticus (Taq) DNA polymerase, methods for reverse transcription polymerase chain reaction (RT-PCR) using said DNA polymerases, and kits comprising said DNA polymerases.

[0003] Reverse transcription polymerase chain reaction (RT-PCR) has evolved as a widely used approach in biotechnology and molecular diagnostics. It represents a powerful tool for amplifying and analysing RNA molecules and has therefore found widespread applications in profiling gene expression, viral detection and the diagnosis of various diseases. Well-established methodologies use viral reverse transcriptases (RTs) to transcribe RNA to cDNA and thermostable DNA polymerases (DNA pols) to amplify the resulting target sequence by PCR.

[0004] The combination of PCR with reverse transcription (RT) laid the basis for various powerful methods in the research field of molecular biotechnology and clinical diagnostics. RT-PCR is used to detect and quantify even low-abundant RNA molecules and is therefore the gold standard in gene expression analysis, generating cDNA libraries and the detection of viral or pathogen infections. Moreover, its excellent potential is applied for both cancer diagnosis and profiling and was recently coupled to high throughput next-generation RNA sequencing (NGS) technologies. In particular, during the global pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), RT-PCR gained strong popularity and the importance of improving established techniques became apparent.

[0005] Classical approaches start with the transcription from RNA to DNA catalysed by viral RTs, followed by DNA purification and real-time PCR. Either fluorescent dyes that intercalate into the DNA product or fluorescently tagged hydrolysis probes (so called TaqMan probes) are used to monitor the reaction. To simplify the procedure, RT and the subsequent DNA amplification were combined in a single tube formulation. This not only saves time, but also reduces the risk of contaminations and eliminates the need to check buffer systems for compatibility. Furthermore, the use of different primer sets with distinctly labelled fluorescence probes enabled the simultaneous targeting of two or even multiple RNA regions in one reaction (multiplexed RT-PCR). This method offers substantial advantages such as saving time, reduced consumption of sample material and lower costs. This technique is successfully used to profile mRNA levels from multiple target genes, detect several viral strains, subtypes or other pathogens simultaneously, and monitor cancer-related biomarkers for personalised medicine approaches. To further enhance the assay performance, the applied enzymes should meet specific requirements. These include high catalytic efficiency at elevated reaction temperatures, good fidelity, enzymatic stability after multiple thermocycling steps, and the ability to produce even longer cDNA products. Taking these criteria into account, both RTs and DNA polymerases have been continuously engineered and improved over the last years.

[0006] However, there is indication that RT and DNA pols can compete for the template binding, which leads to changes in reaction efficiencies that is particularly noticeable with small amounts of input RNA. Compared to the thermostable DNA polymerases, the commonly used viral RTs from the Moloney Murine Leukemia Virus (M-MLV) or the Avian Myeloblastosis Virus (AMV) still have a significant deficit in thermostability. In consequence, the preceding RT reaction is usually conducted at temperatures ranging from 45-60°C, and thus an initial RNA denaturation at high temperatures (around 95°C) is only possible as an additional step prior to the reaction. Higher reaction temperatures are sometimes advantageous because they allow for a more specific priming process, make complex RNA or DNA structures accessible, and inactivate disruptive factors such as RNases.

[0007] Accordingly, the technical problem underlying the present invention is the provision of improved means for RT-PCR providing high reaction efficiency, high sensitivity, and high specificity, as well as a fast and easy assay format. The solution to the above technical problem is achieved by the embodiments characterized in the claims.

[0008] In particular, in a first aspect, the present invention relates to a DNA polymerase derived from wild-type Thermus aquaticus (Taq) DNA polymerase, comprising

[0009] (i) the mutations N483K, E507K, and 1614K;

[0010] (ii) the mutations N483K, S515R, and 1614K;

[0011] (iii) the mutations E507K, and I614K;

[0012] (iv) the mutations L459M, E507K, and 1614K;

[0013] (v) the mutations N483K, E507K, S515R, and 1614K;

[0014] (vi) the mutations L459M, N483K, S515R, and 1614K;

[0015] (vii) the mutations L459M, N483K, E507K, V586G, and 1614K; or

[0016] (viii) the mutations N483K, E507K, S515R, V586G, and 1614K; with regard to the amino acid sequence of wild-type Taq DNA polymerase (SEQ ID NO: 1 ).

[0017] In preferred embodiments, the DNA polymerase of the present invention comprises one of the mutation patterns (i) to (iv) with regard to SEQ ID NO: 1 , preferably, mutation pattern (i) or (ii) with regard to SEQ ID NO: 1 , wherein both of said mutation patterns are particularly preferred.

[0018] Thus, the DNA polymerase of the present invention can comprise or consist of the amino acid sequence as shown in SEQ ID NO: 1 including said mutations. Alternatively, the DNA polymerase of the present invention can comprise or consist of the amino acid sequence corresponding to amino acids 293 to 832 of SEQ ID NO: 1 (known as KlenTaq DNA polymerase) including said mutations, or the corresponding amino acid sequence as shown in SEQ ID NO: 2 including said mutations. Further, the DNA polymerase of the present invention can comprise or consist of the amino acid sequence corresponding to (i) amino acids 4 to 832 of SEQ ID NO: 1 (known as AmpliTaq DNA polymerase), (ii) amino acids 279 to 832 of SEQ ID NO: 1 (known as Klentaql DNA polymerase), (iii) amino acids 290 to 832 of SEQ ID NO: 1 (known as Stoffel fragment), or (iv) amino acids 289 to 832 of SEQ ID NO: 1 (known as TaqA288), including the above mutations.

[0019] The expression “derived from wild-type Taq DNA polymerase” as used herein relates to the fact that the DNA polymerase of the present invention is substantially identical to wild-type Taq DNA polymerase, provided the above mutations are present. However, said expression also includes DNA polymerases whose amino acid sequence has one or more (e.g., 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2) further amino acid substitutions, deletions or additions as compared to the amino acid sequence of wild-type Taq DNA polymerase, provided the above mutations are present and provided the DNA polymerase retains its DNA polymerase activity. Further, the above expression also includes fusion proteins comprising a DNA polymerase of the present invention. In particular, the DNA polymerase of the present invention can comprise an amino acid sequence that has more than 70%, more than 80%, more than 85%, more than 90%, more than 92%, more than 94%, more than 96%, more than 97%, more than 98%, or more than 99% identity to SEQ ID NO: 1 or SEQ ID NO: 2, provided the above mutations are present.

[0020] As indicated above, the amino acid sequence as shown in SEQ ID NO: 2, known as KlenTaq DNA polymerase, corresponds to amino acids 293 to 832 of SEQ ID NO: 1 . More specifically, amino acids 1 to 540 of SEQ ID NO: 2 correspond to amino acids 293 to 832 of SEQ ID NO: 1 , i.e., KlenTaq DNA polymerase is a C-terminal fragment of Taq DNA polymerase.

[0021] The expression “including said mutations” as used herein refers to the fact that the above mutations with regard to the amino acid sequence of wild-type Taq DNA polymerase (SEQ ID NO: 1 ) or KlenTaq DNA polymerase (SEQ ID NO: 2), are present in any case. As an example, the expression “comprising or consisting of the amino acid sequence as shown in SEQ ID NO: 1 including said mutations” as used herein refers to a DNA polymerase comprising or consisting of the amino acid sequence as shown in SEQ ID NO 1 , with the exception that in comparison to the amino acid sequence shown in SEQ ID NO: 1 , the amino acid sequence of the DNA polymerase of the present invention comprises the above mutations. In preferred embodiments, the DNA polymerases of the present invention do not comprise any further mutations other than the mutations indicated herein with regard to SEQ ID NO: 1. In other words, in these embodiments, all positions of SEQ ID NO: 1 other than those for which a particular mutation is defined herein, are as given in SEQ ID NO: 1.

[0022] Means for generating mutations in a known amino acid sequence, as well for generating respective mutated polypeptides such as the DNA polymerase of the present invention, are not particularly limited and are known in the art.

[0023] The notation of mutations as used herein is a standard notation known in the art. As an example, the mutation N483K is a mutation at position 483, where an asparagine (N) has been exchanged for a lysine (K). As a further example the mutation E507K is a mutation at position 507, where glutamic acid (E) has been exchanged for a lysine (K).

[0024] The expression “with regard to SEQ ID NO: 1” as used herein refers to the fact that all respective mutations are to be seen in relation to the wild-type sequence of Taq DNA polymerase provided in SEQ ID NO: 1. As an example, a DNA polymerase according to the present invention can have the amino acid sequence shown in SEQ ID NO: 2 including e.g. the mutations N483K and / or E507K with regard to SEQ ID NO: 1. These mutations are actually in positions 191 and 215 of the actual amino acid sequence of the DNA polymerase. However, said mutations are nevertheless labeled N483K and E507K, since all mutations are to be seen with regard to SEQ ID NO: 1.

[0025] In some embodiments, the DNA polymerase of the present invention further comprises the mutation I707L with regard to SEQ ID NO: 1. Thus, in further preferred embodiments, the DNA polymerase of the present invention comprises

[0026] (ix) the mutations N483K, E507K, 1614K, and I707L;

[0027] (x) the mutations N483K, S515R, 1614K, and I707L; (xi) the mutations E507K, 1614K, and I707L;

[0028] (xii) the mutations L459M, E507K, 1614K, and I707L;

[0029] (xiii) the mutations N483K, E507K, S515R, 1614K, and I707L;

[0030] (xiv) the mutations L459M, N483K, S515R, 1614K, and I707L;

[0031] (xv) the mutations L459M, N483K, E507K, V586G, 1614K, and I707L; or

[0032] (xvi) the mutations N483K, E507K, S515R, V586G, 1614K, and I707L; with regard to SEQ ID NO: 1 , wherein the above mutations patterns (ix) and (x) are particularly preferred. Again, in preferred embodiments, the DNA polymerases of the present invention do not comprise any further mutations other than the mutations indicated herein with regard to SEQ ID NO: 1. In other words, in these embodiments, all positions of SEQ ID NO: 1 other than those for which a particular mutation is defined herein, are as given in SEQ ID NO: 1.

[0033] The term “DNA polymerase” as used herein includes DNA polymerases that have been modified by e.g. natural process such as posttranslational processing, or nonnatural process such as chemical modification. Such modifications can occur on the peptide backbone, amino acid side chains, or the N- or C-terminus. Modifications include e.g. acetylations, acylations, ADP-ribosylations, amidations, covalent attachment of flavines, haem-groups, nucleotides or nucleotide derivatives, lipids or lipid derivatives, cyclizations, disulfide bridges, methylations and demethylations, cystine linkages, formylations, y-carboxylations, glycosylations, hydroxylations, phosphorylations and the tRNA-mediated addition of amino acids.

[0034] Of note, further DNA polymerases derived from Thermus spp. include Thermus filiformis (Tfi) DNA polymerase, Thermus flavus (Tfl) DNA polymerase, N-terminally truncated Tfl DNA polymerase (encompassing residues 280 to 932 of the wild-type Tfl polymerase), Thermus brockianus (Tbr) DNA polymerase, Thermus caldophilus (Tea) DNA polymerase, and Thermus oshimai DNA polymerase (sps17). These DNA polymerases, including the mutations corresponding to the above mutations defined for Tag DNA polymerase, are encompassed herein. In a second aspect, the present invention relates to a DNA polymerase derived from wild-type Thermus aquaticus (Taq) DNA polymerase, comprising the mutations N483K, S515R, 1614K, and M747K with regard to the amino acid sequence of wildtype Taq DNA polymerase (SEQ ID NO: 1 ).

[0035] In other words, when combining the above first and second aspects of the present invention, said invention relates to a DNA polymerase derived from wild-type Thermus aquaticus (Taq) DNA polymerase, comprising

[0036] (i) the mutations N483K, E507K, and 1614K;

[0037] (ii) the mutations N483K, S515R, and 1614K;

[0038] (iii) the mutations E507K, and I614K;

[0039] (iv) the mutations L459M, E507K, and 1614K;

[0040] (v) the mutations N483K, E507K, S515R, and 1614K;

[0041] (vi) the mutations L459M, N483K, S515R, and 1614K;

[0042] (vii) the mutations L459M, N483K, E507K, V586G, and 1614K;

[0043] (viii) the mutations N483K, E507K, S515R, V586G, and 1614K; or

[0044] (xvii) the mutations N483K, S515R, 1614K, and M747K; with regard to the amino acid sequence of wild-type Taq DNA polymerase (SEQ ID NO: 1 ).

[0045] According to the above second aspect of the present invention, it is preferred that the DNA polymerase of the present invention comprises the mutation pattern (xvii) with regard to SEQ ID NO: 1 .

[0046] According to the second aspect of the present invention, as well as for the combined first and second aspect of the present invention, all relevant definitions and limitations defined for the first aspect of the present invention equally apply.

[0047] In particular, the DNA polymerase of the present invention according to the second aspect can comprise or consist of the amino acid sequence as shown in SEQ ID NO: 1 including said mutations. Alternatively, the DNA polymerase of the present invention according to the second aspect can comprise or consist of the amino acid sequence corresponding to amino acids 293 to 832 of SEQ ID NO: 1 (known as KlenTaq DNA polymerase) including said mutations, or the corresponding amino acid sequence as shown in SEQ ID NO: 2 including said mutations. Further, the DNA polymerase of the present invention according to the second aspect can comprise or consist of the amino acid sequence corresponding to (i) amino acids 4 to 832 of SEQ ID NO: 1 (known as AmpliTaq DNA polymerase), (ii) amino acids 279 to 832 of SEQ ID NO: 1 (known as Klentaql DNA polymerase), (iii) amino acids 290 to 832 of SEQ ID NO: 1 (known as Stoffel fragment), or (iv) amino acids 289 to 832 of SEQ ID NO: 1 (known as TaqA288), including the above mutations.

[0048] The expressions / terms “derived from wild-type Taq DNA polymerase”, “including said mutations”, “with regard to SEQ ID NO: 1”, and “DNA polymerase” are as defined above for the first aspect of the present invention.

[0049] In some embodiments, the DNA polymerase according to the second aspect of the present invention further comprises the mutation I707L with regard to SEQ ID NO: 1 . Thus, in further preferred embodiments, the DNA polymerase according to the second aspect of the present invention comprises the mutations N483K, S515R, I614K, I707L, and M747K.

[0050] In this context, further DNA polymerases derived from Thermus spp., including Thermus filiformis (Tfi) DNA polymerase, Thermus flavus (Tfl) DNA polymerase, N- terminally truncated Tfl DNA polymerase (encompassing residues 280 to 932 of the wild-type Tfl polymerase), Thermus brockianus (Tbr) DNA polymerase, Thermus caldophilus (Tea) DNA polymerase, and Thermus oshimai DNA polymerase (sps17), including the mutations corresponding to the above mutations defined for Taq DNA polymerase, are encompassed in the second aspect of the present invention.

[0051] In preferred embodiments, the DNA polymerase of the present invention according to the second aspect does not comprise any further mutations other than the mutations indicated herein with regard to SEQ ID NO: 1. In other words, in these embodiments, all positions of SEQ ID NO: 1 other than those for which a particular mutation is defined herein, are as given in SEQ ID NO: 1.

[0052] In a further aspect, the present invention relates to a nucleic acid comprising a nucleotide sequence coding for a DNA polymerase according to the present invention.

[0053] In a further aspect, the present invention relates to a vector comprising a nucleic acid according to the present invention. The term “vector” as used herein relates to any vehicle for the transportation of a nucleic acid into a cell. In particular, said term includes plasmid vectors, viral vectors, cosmid vectors, and artificial chromosomes, wherein plasmid vectors are particularly preferred. Preferably, plasmid vectors are suitable for expression of the DNA polymerases of the present invention in a prokaryotic or eukaryotic cell. Respective plasmid vectors are known in the art.

[0054] In a further aspect, the present invention relates to a host cell comprising the vector and / or the nucleic acid of the present invention. Suitable host cells that can be used for the recombinant expression of the DNA polymerases of the present invention are not particularly limited and are known in the art. They include for example suitable bacterial cells, yeast cells, plant cells, insect cells and mammalian cells.

[0055] In a yet further aspect, the present invention relates to a reverse transcription polymerase chain reaction (RT-PCR) method, comprising the steps:

[0056] (a) reverse transcription of one or more RNA(s) of interest to cDNA, and

[0057] (b) amplification of the cDNA generated in step (a) by PCR, wherein both of steps (a) and (b) are catalyzed using a DNA polymerase according to the first aspect of the present invention.

[0058] The present invention according to this aspect encompasses any methods in which reverse transcription of one or more RNA(s) of interest into cDNA, and the amplification of said cDNA by PCR, is of interest. This includes e.g. quantitative PCR methods, such as e.g. RT-qPCR. Further, as demonstrated herein, respective methods include multiplex methods (e.g. duplex, triplex, quadruplex, and multiplex methods), i.e., methods in which two, three, four, or more different RNAs of interest are assayed.

[0059] Preferably, in the methods of the present invention, both of steps (a) and (b) are performed concurrently and in the same reaction vessel. Further, it is preferred that no other enzyme using a nucleic acid as substrate is present during steps (a) and (b).

[0060] In related aspects, the present invention relates to (i) a method for the reverse transcription of one or more RNA(s) of interest to cDNA, wherein said step is catalyzed using a DNA polymerase according to the first aspect of the present invention, and (ii) a method for the amplification of cDNA by PCR, wherein said step is catalyzed using a DNA polymerase according to the first aspect of the present invention.

[0061] In a yet further aspect, the present invention relates to a reverse transcription polymerase chain reaction (RT-PCR) method, comprising the steps:

[0062] (a) reverse transcription of one or more RNA(s) of interest to cDNA, and

[0063] (b) amplification of the cDNA generated in step (a) by PCR, wherein step (a) is catalyzed using a DNA polymerase according to the second aspect of the present invention.

[0064] In preferred embodiments, step (b) of the above method is catalyzed by a second DNA polymerase, i.e., a DNA polymerase that is different from the DNA polymerase according to the second aspect of the present invention. Suitable DNA polymerases are not particularly limited and are known in the art. Such DNA polymerases include wild-type Taq DNA polymerase, Thermus brockianus (Tbr) DNA polymerase, Thermus caldophilus (Tea) DNA polymerase, Thermus filiformis (Tfi) DNA polymerase, Thermus flavus (Tfl) DNA polymerase, Thermococcus fumicolans (Tfu) DNA polymerase, Thermococcus gorgonarius (Tgo) DNA polymerase, Thermococcus litoralis (Tli) DNA polymerase, Pyrococcus furiosus (Pfu) DNA polymerase, Pyrococcus woesei (Pwo) DNA polymerase, Vent DNA polymerase, Deep Vent DNA polymerase, Thermococcus kodakarensis (KOD) DNA polymerase, Phusion DNA polymerase, and Q5 DNA polymerase, wherein Taq DNA polymerase is preferred.

[0065] In alternative embodiments, both of steps (a) and (b) of the above method are catalyzed using a DNA polymerase according to the second aspect of the present invention. In such embodiments, it is preferred that no other enzyme using a nucleic acid as substrate is present during steps (a) and (b).

[0066] The present invention according to this aspect encompasses any methods in which reverse transcription of one or more RNA(s) of interest into cDNA, and the amplification of said cDNA by PCR, is of interest. This includes e.g. quantitative PCR methods, such as e.g. RT-qPCR. Thus, the PCR can be quantitative PCR (qPCR). Further, the PCR can be digital PCR (dPCR).

[0067] Furthermore, respective methods include multiplex methods (e.g. duplex, triplex, quadruplex, and multiplex methods), i.e., methods in which two, three, four, or more different RNAs of interest are assayed.

[0068] In some embodiments, it is preferred that both of steps (a) and (b) are performed concurrently and in the same reaction vessel.

[0069] In related aspects, the present invention relates to (i) a method for the reverse transcription of one or more RNA(s) of interest to cDNA, wherein said step is catalyzed using a DNA polymerase according to the second aspect of the present invention.

[0070] In a final aspect, the present invention relates to a kit comprising a DNA polymerase according to the first or second aspect of the present invention. In preferred embodiments, the kit of the present invention further comprises suitable buffers and / or suitable disposables. As used herein, the term “comprising” / ”comprises” expressly includes the terms “consisting essentially of” / ” consists essentially of” and “consisting of” / ” consists of’, i.e., all of said terms are interchangeable with each other herein.

[0071] Further, as used herein, the term “about” refers to a modifier of the specified value of ± 10%, preferably ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2%, ± 1 %, ± 0.5%, or ± 0.1 %. Thus, by way of example the term “about 100” as used herein can refer to ranges of 90 to 110, 91 to 109, 92 to 108, 93 to 107, 94 to 106, 95 to 105, 96 to 104, 97 to 103, 98 to 102, 99 to 101 , 99.5 to 100.5, or 99.9 to 100.1.

[0072] The present invention relates to the development of novel Thermus aquaticus DNA polymerase I (Taq pol) variants that each are able to catalyse both steps of (i) transcribing RNA to cDNA and (ii) amplifying the resulting target sequence by PCR simultaneously in a single tube without the need of viral RTs. In combination with their excellent thermostability (up to 95°C), the novel Taq pol variants of the present invention are suitable for employment in dye- or probe-based RNA detection methods. Moreover, the herein reported Taq pol variants are the first DNA pols that are capable of performing multiplex detection of various RNA targets in a single tube with a single enzyme. Thus, discovery marks a significant advancement of current RT-PCR approaches and contributes to simplifying and reducing costs in molecular diagnostics.

[0073] Thus, the present invention reports the development and application of novel DNA polymerase variants that are able to catalyse RT and DNA amplification simultaneously without the need of viral RTs, addition of manganese ions or to be fused to other enzyme scaffolds. The enzymes are derived from the DNA polymerase I of the thermophilic bacterial strain Thermus aquaticus (Taq) and are therefore characterised by their excellent thermostability, high processivity, and ability to digest TaqMan probes. The enzymes were obtained by combinatorically investigating two independently discovered mutation pools that enhance the RT activity of the N-te rm inally truncated form of Taq pol termed KlenTaq (KTq) DNA polymerase. Promising variants were selected after screening the mutant library for RT and PCR activity. It could be shown that the application of those enzymes in RT- PCR further simplifies the one-tube formulation by eliminating the need for viral RTs as a second enzyme. In this way, it was possible to perform RT-PCR by using either SYBR® green I as intercalating fluorescent dye or a TaqMan probe based approach. Moreover, it was possible to detect up to four RNA targets simultaneously with a detection limit of 20 copies. This method marks a significant advancement, enabling the simultaneous detection of four RNA targets through quadruplex RT-PCR, all achieved through the utilization of a single enzyme capable of catalysing both RT and DNA amplification.

[0074] Moreover, reverse transcriptases are important tools in molecular biology. One disadvantage of commonly used viral AMV and M-MLV enzymes is low thermal stability and robustness against inhibitors. In previous studies, thermostable DNA polymerases like Taq polymerase have been engineered to show increased reverse transcriptase activity, while maintaining DNA polymerase activity.

[0075] Thus, the present invention further describes a mutated Taq polymerase that has superior RT activity, but mostly lost DNA polymerase activity and therefore can be used as a heat-stable and inhibitor-tolerant standalone RT in combination with established DNA polymerases.

[0076] The figures show:

[0077] Crystal structures of RT-KTq / Mut RT and Taq DNA pol.

[0078] A) The overall crystal structure of RT-KTq and Mut_RT DNA pol is shown as cartoon (adapted from PDB ID: 4BWM using PyMOL (Schrodinger, LLC, New York, NY)). Amino acid mutations specific for RT-KTq are coloured in blue and mutations specific for Mut_RT are coloured in green (side chains depicted as sticks). The KTq pol backbone is coloured in light grey. B) The overall crystal structure of Taq pol is shown as cartoon (adapted from PDB ID: 1TAQ using PyMOL (Schrodinger, LLC, New York, NY)). The KTq pol backbone is coloured in light grey and the 5’ -3’ nuclease domain is depicted in dark grey. The amino acids that are changed to form the RT-Taq are coloured in blue. C) Architecture of the KTq pol variants (top) and Taq pol (bottom). The positions of amino acid mutations are indicated in blue (RT- KTq, RT-Taq) and green (Mut_RT). D) Combination strategy of the new Taq pol enzyme library. All eight amino acid mutations were combined to generate the wildtype Taq pol (without mutations), all single, double, triple and 4-8 fold mutants giving 256 Taq pol variants in total. The plasmid mix was transformed into E. coli cells, plated and 2660 single colonies were picked to form the library.

[0079] Comparison of RT-KTq, Mut RT and all single mutants of Mut RT regarding linear RT- and PCR activity.

[0080] A) Primer extension with radioactively labelled primer from the artificial RNA oligonucleotide. Reaction mix contained 2 nM KTq variant (as indicated), 200 pM dNTPs (each), 150 nM labelled primer and 225 nM RNA template. Reaction was conducted for 10 and 30 min. Control lane (con.) depicts the signal for the primer only. Analysis was performed by 12% denaturing PAGE and phosphor imaging. B) Amplification curves after RT-PCR from the artificial RNA oligonucleotide. Reaction mix contained 100 pM template, 100 nM forward and reverse primer, 200 pM dNTPs (each), 100 nM KTq pol variant (as indicated) and 1x SYBR® green I. The control Mix (con.) contained the same mastermix, but without RNA template. RT-PCR were performed in duplicates. The mean of Cq values are depicted in the table. C) Analysis of product formation by agarose gel electrophoresis (2.5% gel).

[0081] Figure 3:

[0082] Screening approach and selection for identifying Tag pol variants with increased RT-PCR activity.

[0083] A) Principle of the screening assay. RT and amplification of the cDNA is catalysed by different Taq pol variants. The applied TaqMan probe is 5'- fluorescently labelled with FAM (6-carboxyfluorescein) and 3'- labelled with BHQ1 (Black Hole Quencher 1 ). Product formation can be followed by monitoring the increase in fluorescence signal as the probe is digested by the Taq pol variants and fluorescence is no longer quenched by BHQ1. B) Selection of promising Taq pol variants are shown as an example for the first expression plate (amplification curve for lysate containing the RT-Taq depicted in red and amplification curve for lysates containing different Taq pol variants depicted in blue). Selection of promising variants were based on lower Cq values than those of the RT-Taq (red curve) and amplification curves following a sigmoidal shape. C) Frequency of each mutation (L459M, N483K, E507K, S515R, V586G, 1614K, I638F and M747K) is depicted in active vs. inactive Taq pol variants. Left: 15 different Taq pol variants were included in the group of active variants and right: 8 Taq pol variants were included in the group of inactive variants. B) Mutation level (O-fold, 1 -fold, 2-fold... ) is plotted against their frequency. Left: 15 different Taq pol variants were included in the group of active variants and right: 8 Taq pol variants were included in the group of inactive variants.

[0084] Figure 4:

[0085] Determination of suitable screening conditions by conducting RT-PCR from SARS- CoV-2 RNA with RT-Tag.

[0086] A) Purified RT-Taq (concentrations as indicated), 150 nM Taq pol aptamer and 1000 c / pl RNA was present in the reaction mix. A temperature gradient was used to find the suitable annealing temperature of 61 °C. B) Amplification of the RT-PCR was catalysed by cell lysates containing the RT-Taq enzyme (lysate volume as indicated). Left: Appropriate lysate volume was determined to be 3 pl, middle: 800 nM Taq pol aptamer was determined to be suitable for screening, and left: 1000 c / pl RNA was found to be sufficient to monitor the reaction as the amount of RNA template has to be kept low in view of the size of the screening. The reactions were performed only once. 20 best lysates.

[0087] 3 pl lysate, 800 nM Taq pol aptamer, 670 nM primer, 170 nM TaqMan probe and SARS-CoV-2 RNA (concentrations as indicated) were present in the reaction mix. Fwd primer was varied to generate PCR products with different lengths (as indicated). The same mastermix was used for the control reaction (con.), but the amount of RNA template was replaced by water. Of note, lysate F2 was only tested for 5 conditions. The reactions with 1000 c / pL template and 72 bp amplicon size were performed twice and other experiments were conducted only once.

[0088] Figure 6:

[0089] SDS-PAGE analysis of cell lysates containing Tag pol variants.

[0090] A) Lysates from the 20 most promising variants are analysed. B) Lysates from the inactive variants were analysed. variants.

[0091] 30 nM enzyme, 100 nM Tag pol aptamer, 670 nM primer, 170 nM TagMan probe and SARS-CoV-2 RNA (concentrations as indicated) were present in the reaction mix. Fwd primer was varied to generate PCR products with different lengths (as indicated). The same mastermix was used for the control reaction (con.), but the amount of RNA template was replaced by Milli-Q water. The reactions with diluted RNA template were performed twice and reactions forming longer amplicons were conducted once.

[0092] Figure 8: curves after RT-PCR from SARS-CoV-2 RNA and from the artificial

[0093] RNA used in preliminary studies.

[0094] A) 1000 c / pL SARS-CoV-2 RNA was used as template and different forward primers were applied to generate PCR products of different lengths (as indicated). 30 nM purified Tag pol variants (as indicated) and 100 nM Tag pol aptamer was used for catalysis. The reaction was monitored by SYBR® green I intercalating into the forming cDNA. B) 100 nM artificial RNA template was present in the reaction. 100 nM purified Tag pol variants (as indicated) and 100 nM Tag pol aptamer was used for catalysis. The reaction was monitored by SYBR® green I intercalating into the forming cDNA. A) / B) For the control reaction (con.), the same mastermix was used but the amount of RNA template was replaced by Milli-Q water. The reactions from the SARS-CoV-2 RNA template were performed twice and reactions from the artificial RNA template were conducted three times.

[0095] Figure 9: and

[0096] A) 10.000 c / pL SARS-CoV-2 RNA was used as template and the genes nCoV_N1 , N2 and E were analysed independently (as indicated). 120 nM purified Taq pol variants (as indicated) and 400 nM Taq pol aptamer was used for catalysis. 670 nM primer, 170 nM TaqMan probe (in case of nCoV_N1 / N2) or 850 nM primer, 216 nM TaqMan probe (in case of nCov_E) were present in the reaction mix. Reactions were monitored by measuring the fluorescence from the 6-FAM dye (nCoV_N1 , blue curves), the Sun dye (nCoV_N2, green curves) and the Texas Red dye (nCov_E, purple curves). B) 10.000 c / pL SARS-CoV-2 RNA was used as template and the genes nCoV_N1 , N2 and E were analysed simultaneously (triplex RT-PCR). The reaction conditions were similar to reactions described in A), but all primer sets were present in the same reaction. C) SARS-CoV-2 RNA template was diluted stepwise and the genes nCoV_N1 , N2 and E were analysed independently (singleplex). D) SARS-CoV-2 RNA template was diluted stepwise and the genes nCoV_N1 , N2 and E were analysed simultaneously (triplex RT-PCR). C) / D) The same reaction conditions were use as in A) or B). Respective Cq values were plotted against the decadic logarithm of the template concentration. Linear regression was applied. Linear functions, R2 values and the PCR efficiencies are indicated in Table 4. Error bars indicating the standard deviation of duplicates are shown in black. A) / B) / C) / D) The same mastermix was used for the control reaction (con.), but the amount of RNA template was replaced by water. Reactions were performed at least in duplicates. Fiqure 10: curves after multiplex RT-PCR from SARS-CoV-2 RNA and human

[0097] RNase P

[0098] A) 10.000 c / L SARS-CoV-2 RNA was used as template and the genes nCoV_N1 , N2 and E were analysed simultaneously. 1 ng / pL universal human reference RNA was used to monitor amplification from the RNase P transcript. 120 nM purified Taq pol variants (as indicated) and 400 nM Taq pol aptamer was used for catalysis. 670 nM primer, 170 nM TaqMan probe (in case of nCoV_N1 / N2, RNase P) or 850 nM primer, 216 nM TaqMan probe (in case of nCov_E) were present in the reaction mix. Reactions were monitored by measuring the fluorescence from the 6-FAM dye (nCoV_N1 , blue curves), the Sun dye (nCoV_N2, green curves), the Texas Red dye (nCov_E, purple curves) and the Cy5 dye (RNase P, orange curves). B) SARS-CoV- 2 RNA template was diluted stepwise while keeping the human reference RNA constant. The genes nCoV_N1 , N2, E and RNase P transcript were analysed simultaneously (quadruplex RT-PCR). Respective Cq values were plotted against the decadic logarithm of the template concentration. Linear regression was applied. Linear functions, R2 values and the PCR efficiencies are indicated in Table 4. Error bars bars indicating the standard deviation of duplicates are shown in black. A) / B) The same mastermix was used for the control reaction (con.), but the amount of RNA template was replaced by water. Reactions were performed at least in duplicates.

[0099] Fiqure 11 :

[0100] Investigation of the polymerase fidelity by NGS Error rates from RT-Taq, Mut_RT, Taq pol V2, V3, V2 IL processing the SARS-CoV-2 RNA in the N1 region are illustrated (shown is the area of the amplicon without primer binding sites). 30 nM Taq pol variant and 100 nM Taq pol aptamer was used for RT-PCR.

[0101] 12:

[0102] Error distribution of Taq pol variants while processing the SARS-CoV-S RNA in the N1 region. Error rates (black dots) for each DNA pol were grouped separately according to their base identity and illustrated in a dot plot (errors from region without primer binding sites are included). Grey lines indicate the mean of each group and the range of the data points. Dot plots were prepared by using GraphPad Prism version 6.00 for Windows, GraphPad Software, La Jolla California USA.

[0103] Figure 13:

[0104] Comparison of RT-Taq, RT-KTq and Mut_RT regarding linear RT- and PCR activity. A) Primer extension with radioactively labeled primer and the unmodified oligonucleotide from the MJ-screening. Reaction mix contained 2 nM Taq pol variant (as indicated), 200 pM dNTPs (each), 150 nM labeled primer and 225 nM RNA template. Reaction time was 10 and 30 min. Control lane (con.) depicts the signal for the primer only. Analysis was performed by 12% denaturing PAGE and phosphor imaging. B) Amplification curves after RT-PCR from the unmodified RNA oligonucleotide used in the MJ-screening. Reaction mix contained 100 pM RNA template, 100 nM forward and reverse primer, 200 pM dNTPs (each), 100 nM Taq pol variant (as indicated) and 1x SYBR green I. Control reaction contained the same mastermix, but without RNA template. Cq values for each replicate and mean Cq values are depicted in the table. Reactions were performed in duplicates. C) Analysis of product formation by agarose gel electrophoresis (2.5% gel). RT-KTq (KTq pol L459M, S515R, I638F and M747K), RT-Taq (Taq pol L459M, S515R, I638F and M747K) and Mut_RT (KTq pol N483K, E507K, K540Y, V586G, 1614K).

[0105] Figure 14:

[0106] Mutant Taq polymerases were used in the presence (blue bars) or absence of wildtype Taq polymerase (grey bars) to reverse transcribe 10.000 copies per reaction of a SARS COV2 N1 in vitro transcript. A Quantinova Multiplex PCR reaction mix w / o Taq polymerase was used and supplemented with 2.5U per reaction of the corresponding polymerases. Amplification was performed on a BioRad CFX 384 instrument and cycling conditions shown in Table 9. Mutants V2 (N483K E507K 1614K) and V3 (N483K S515R 1614K) showed little difference between standalone and Taq supplemented, confirming good RT and DNA polymerase activity. Mutant V6 (N483K S515R 1614K M747K) showed a high Ct value if used as sole enzyme, but the lowest Ct of all tested variants in the presence of a wildtype Taq polymerase.

[0107] Figure 15:

[0108] Amplification plot of SARS COV2 N1 amplifications. Red: Reference One-Step RT PCR Quantinova Multiplex RT PCR Kit. Blue: Mutant V6 combined with wildtype Taq. Green: Mutant V6 alone.

[0109] Figure 16:

[0110] Mutant V6 sensitivity compared to mutant V3.

[0111] Figure 17:

[0112] QuantiNova Multiplex PCR mastermix w / o Taq polymerase was complemented with mutant V6 and the primer complementary to the SARS CoV2 N1 in vitro transcript only, or both primers. As expected, with just one long incubation at 65°C after initial denaturation, there is no difference between one and two primer setups. The enzyme performs a single conversion of RNA into cDNA, like a conventional RT enzyme. With increasing cycles, Cts decrease at a rate indicating linear amplification of RNA into cDNA with only one primer present. With both primers, Ct differences indicate exponential amplification, thus cDNA synthesis and DNA amplification happening simultaneously.

[0113] Figure 18:

[0114] Comparison of mutant Taq (V6) and QIAcuity OneStep Advanced Probe using a cycling protocol with an initial denaturation at 95°C for 30sec.

[0115] Figure 19:

[0116] QuantiNova Multiplex PCR mastermix with Taq polymerase was complemented with mutant V6 or V3 in ratios 1 : 1 (2.5LI each) up to 1 : 10 (25U mutant Taq). No significant differences in Ct values were observed, indicating the absence of inhibition of wildtype Taq polymerase by the mutant enzymes. Figure 20:

[0117] Tolerance towards hematin was tested in a range of 20 - 200pM. Internal control results are shown.

[0118] Figure 21 :

[0119] Inhibitor tolerance of the reference mix QIAcuity OneStep Advanced Probe Mix. Reaction setup and PCR was conducted as per handbook recommendations. Only ACTB resulted in minimal residual guantification at the lowest inhibitor concentration, all other reactions were fully inhibited.

[0120] Figure 22:

[0121] The ability to form primer dimer artefacts was tested. Mutant Tag (V6) was incubated with dimer forming oligonucleotides for 17 hours at room temperature. Results show that Tag specific aptamer, antibody, as well as hot start dNTPs (Trilink CleanAmp) all significantly reduce the residual activity. Combinations of hot start methods lead to further reduction, all combined finally result in a delta Ct of 15.9.

[0122] The present invention relates to the following amino acid and nucleotide seguences. Additional nucleotide seguences are indicated in Table 1.

[0123] SEQ ID NO: 1

[0124] (amino acid seguence of wild-type Thermus aquaticus DNA polymerase)

[0125] MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSLLKALKEDGD AVIWFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADD VLASLAKKAEKEGYEVRILTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWA DYRALTGDESDNLPGVKGIGEKTARKLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLK LSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWP PPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLA LREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRL EGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGH PFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTK LKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIA

[0126] EEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRR

[0127] AAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYV

[0128] ETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARML

[0129] LQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKE

[0130] SEQ ID NO: 2

[0131] (amino acid sequence of a C-terminal fragment of wild-type Thermus aquaticus DNA polymerase; KlenTaq DNA polymerase):

[0132] ALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLL

[0133] AKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERL

[0134] FANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEA

[0135] EVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKI

[0136] LQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQ

[0137] RIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPRE

[0138] AVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLE

[0139] EGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRL

[0140] EEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKE

[0141] (SEQ ID NOs: 3 to 45 shown in Table 1 )

[0142] The present invention will be further illustrated by the following examples without being limited thereto.

[0143] Examples

[0144] Material and methods: Oligonucleotides

[0145] DNA oligonucleotides were ordered from IDT or Biomers in HPLC grade and directly used in RT-PCR assays and primer extension reactions. SARS-CoV-2 RNA was purchased from the European Commission Joint Research Centre (CRM code: EURM-019) and were directly used. For primer extension reactions analysed by denaturing PAGE, oligonucleotides were purified by preparative PAGE prior to usage. Thereafter, radioactive labelling with [y-32p]-ATP (Hartmann Analytic) and T4 polynucleotide kinase (NEB) was conducted following the manufacturer’s protocol. The concentration of the used oligonucleotides was determined by measuring the absorbance at 260 nm and applying the Lambert-Beer law. Used dNTPs were supplied by Jena Bioscience. DNA and RNA sequences of all oligonucleotides are shown in Table 1 .

[0146] Table 1 : RNA / DNA oligonucleotides using in this study

[0147] Preliminary experiments using parental DNA polymerases

[0148] Introducing I707L mutation

[0149] Multiplex RT-PCR

[0150] $ 5'-radioactively labelled in case of primer extension experiments

[0151] * Nucleotides marked as “N” are random nucleotides used as indices (Unique molecular identifier = UMI)

[0152] Taq DNA pol library preparation

[0153] 256 pGDR11 plasmids carrying different Taq pol genes were purchased from Genewiz (Table 2) and delivered in one single tube. Transformation of electro competent E. coli BL21 DE3 (Stratagene, Table 3) cells was performed by adding 10 ng plasmids to 100 pL cells and applying an electric pulse with 1800 V, 200 O and a pulse length of around 5 ms by Gene Pulser Xcell (Bio-Rad). Cells were mixed with 1 mL SOC medium and incubated at 37°C for 1 h. After plating and incubation of the cells (37°C, overnight), 100 pL of LB medium containing 100 pg / mL carben icil lin disodium salt was inoculated with a single cell colony to end up with 7x 384 deep-well plates. Cells were grown at 37°C overnight, 100 pL glycerol was added in each well and library plates were stored at -80°C.

[0154] Table 2: Plasmids used in this study Table 3: E. coli strains used in this study

[0155] Generation of E. coli cell lysates in 96 well plates

[0156] 1 mL LB medium with 100 pg / mL carbenicillin disodium salt was inoculated with glycerol stocks of E. coli and cells were grown at 37°C in a shaker. After reaching an ODeoo of 0.6, expression was induced by the addition of IPTG (0.4 mM final concentration). Cells were incubated at 37°C in a shaker for 3 h and then harvested by centrifugation with 4000 rpm at 4°C for 30 min. Pellets were resuspended and lysed by the addition of 800 pL 1x lysis buffer I (50 mM Tris-HCI (pH 9.2), 16 mM (NH4)2SO4, 2.5 mM MgCL, 0.1 % (v / v) Tween 20, 0.5 mg / mL lysozyme) and incubation at 37°C for 20 min. After denaturing of E. coli host proteins at 75°C for 45 min, plates were centrifuged at 4000 rpm and 4°C for 30 min and stored at 4°C. Uniform expression was exemplary analysed by SDS-PAGE.

[0157] Large scale protein production and purification

[0158] 400 mL LB medium with 100 pg / mL carbenicillin disodium salt was inoculated with 4 mL of the respective E. coli overnight culture and incubated at 37°C in a shaker. After reaching an ODeoo of 0.6-0.8, induction was performed by adding 1 mM IPTG. Cells were incubated at 37°C in a shaker for 4 h. Then, cultures were harvested by centrifugation at 4°C and 4000 rpm for 30 min and pellets were stored at -20°C. Cell pellets were resuspended and lysed in 10 mL 1x lysis buffer II (10 mM Tris-HCI (pH 9.2), 300 mM NaCI, 2.5 mM MgCL, 0.1 % (v / v) Triton X-100, 1 mg / mL lysozyme) at 37°C for 20 min. After heat inactivating of E. coli host proteins at 75°C for 45 min, lysates were ultra-centrifuged at 20.000 rpm for 1 h. 5 mM imidazole was added to the supernatant and metal ion-based affinity purification was conducted by applying the complete™ His-Tag purification resin (Roche). Here, 3 mL beads were washed 4 times with 5 mL calibration buffer (10 mM Tris-HCI (pH 9.2), 300 mM NaCI, 2.5 mM MgCl2, 0.1 % (v / v) Triton X-100, 5 mM imidazole) by alternate mixing and centrifugation at 1000 rpm and 4°C for 3 min. The cell lysate was mixed with the calibrated beads and shaken at 4°C in an overhead shaker overnight. The protein bead solution was applied onto a 15 mL chromatography column and the flow through was collected. Then, beads were washed 4 times with 5 mL washing buffer (10 mM Tris-HCI (pH 9.2), 300 mM NaCI, 2.5 mM MgCL, 0.1 % (v / v) Triton X-100, 20 mM imidazole). Protein elution was performed by loading 1.6 mL elution buffer I (100 mM Tris-HCI (pH 9.2), 5 mM MgCL, 200 mM imidazole) 6 times and collecting the flow through separately at 4°C. Protein purification was analysed by SDS-PAGE. Pure enzyme fractions were combined and concentrated by using an Amicon filter tube (Amicon Ultra-15, 30 kDa MWCO, Millipore) at 4°C and 4000 rpm. Remaining imidazole was removed by washing the protein solution 4 times with 20 mL elution buffer II (100 mM Tris-HCI (pH 9.2), 5 mM MgCL) followed by a final concentration step to obtain an end volume of 0.4-0.5 mL. For storage, 20x storage buffer (100 mM Tris-HCI (pH 9.2), 320 mM (NH^SCM, 5 mM MgCL, 2% (v / v) Tween 20) was added in an amount equal to 1 / 9th of the volume of the protein (final 2x storage buffer). Finally, autoclaved glycerol was added to reach a final concentration of 50% and enzyme stocks were stored in aliquots at -20°C. Protein concentration was determined by employing a Bradford assay (Roti®Quant, Roth) by using BSA as reference protein.

[0159] Primer extension with radioactively labelled primer

[0160] Reactions were carried out in 10 pL end volume. The reaction mixture contained 225 nM RNA oligonucleotide template, 150 nM radioactively labelled DNA primer, 100 pM of dNTPs (each) and 2 nM KTq pol variant in 1x Taq reaction buffer (50 mM Tris-HCI (pH 9.2), 16 mM (NH^SCM, 2.5 mM MgCL, 0.1 % (v / v) Tween 20). First, template and primer were annealed by heating the mixture to 95°C for 2 min and then cooling it down stepwise to 4°C. Then, dNTPs in 1x Taq reaction buffer was added and pre-warmed to 55°C. Primer extension was started by the addition of 4 pL KTq pol variant in 1x Taq reaction buffer. Reactions were allowed to proceed at 55°C for indicated reaction times and were quenched by the addition of 20 pL stop solution (80% formamide, 20 mM EDTA, 0.25% (w / v) bromophenol blue, 0.25% (w / v) xylene cyanole). After denaturation at 95°C for 5 min, reactions were analysed by 12% denaturing PAGE and visualised by phosphor imaging conducted on a TyphoonTM FLA 9500 (GE Healthcare Life Science). Reactions were repeated twice.

[0161] RT-PCR assay using the RNA oligonucleotide as template

[0162] RT-PCRs were performed in a reaction volume of 10 pL containing 100 pM RNA oligonucleotide as template, 100 nM forward and reverse primer (each), 200 pM dNTPs (each), 0.5 M betaine, 1x SYBR® green I, 100 nM Taq pol aptamer and 100 nM of Taq / KTq pol variants in 1x Taq reaction buffer (50 mM Tris-HCI (pH 9.2), 16 mM (NH4)2SO4, 2.5 mM MgCL, 0.1 % (v / v) Tween 20). RT-PCR was conducted following the thermocycling protocol: 95°C for 60s, then 40 cycles of 95°C for 15 s and 62°C for 30 s. Fluorescence intensities were measured after each of the 40 cycles using the Lightcycler® 96 instrument (Roche). Reactions were performed two or three times.

[0163] Screening assay for Taq pol variants with increased RT-PCR activity

[0164] For screening, 10 pL reaction mixture contained 250 pM dNTPs (each), 670 nM forward and reverse primer (each), 0.5 M betaine, 800 nM Taq pol aptamer, 3 pL of the respective E. coli cell lysate, 170 nM fluorescent probe and SARS-CoV-2 RNA template as indicated in 1x Taq reaction buffer (50 mM Tris-HCI (pH 9.2), 16 mM (NH4)2SO4, 2.5 mM MgCL, 0.1 % (v / v) Tween 20). After preincubation at 61 °C for 150 s, 10 cycles with 95°C for 5 s and 61 °C for 150 s were carried out. Then, 45 cycles with 95°C for10 s and 57°C for 35 s followed. Fluorescent intensities were measured after each of the 45 cycles. PCR was monitored by using the CFX384 Touch Real-Time PCR Detection System (Biorad). Reactions were performed in 384 well plates, thus 4 lysate plates (96 wells) could be analysed in parallel. For easy handling, pipetting was conducted by using the pipetting robot ASSIST plus from Integra. Enzyme performance was evaluated by assessing the Cq values of each Taq pol variant and the shape of the product curve. Product formation was exemplary analysed by agarose gel electrophoresis (2.5% agarose gel in 1x TAE buffer).

[0165] RT-PCR using SARS-CoV-2 RNA as template and purified Taq pol variants

[0166] For reactions with purified Taq pol variants, 10 pL reaction mixture contained 250 pM dNTPs (each), 670 nM forward and reverse primer (each), 0.5 M betaine, 100 nM Taq pol aptamer, 30 nM respective Taq pol variant, 170 nM fluorescent probe or 1x SYBR® green I and SARS-CoV-2 RNA template as indicated in 1x Taq reaction buffer (50 mM Tris-HCI (pH 9.2), 16 mM (NH^SCM, 2.5 mM MgCI2, 0.1 % (v / v) Tween 20). After preincubation at 61 °C for 150 s, 10 cycles with 95°C for 5 s and 61 °C for 150 s were carried out. Then, 35 cycles with 95°C for 10 s and 57°C for 35 s followed. Fluorescent intensities were measured after each of the 35 cycles. PCR was monitored by using the CFX384 Touch Real-Time PCR Detection System (Biorad).

[0167] Site directed mutagenesis for introducing I707L mutation

[0168] For PCR, Phusion High-Fidelity DNA Polymerase (NEB) was used according to the manufacturer's protocol. The reaction mixture contained 200 pM dNTPs (each), 0.5 pM forward primer (which had the desired point mutation in its DNA-strand), 0.5 pM reverse primer (with a phosphate residue on its 5'-ending), 2 ng / pL plasmid DNA, 3% (v / v) DMSO (NEB) and 0.02 U / pL Phusion DNA Polymerase (NEB) in 1x Phusion HF buffer (NEB). The reaction had a total volume of 20 pL and was performed in triplicates. An initial denaturation step was performed at 98°C for 30s, followed by 30 PCR cycles. One cycle consisted of a denaturation step at 98°C for 10 s, an annealing step at 60°C for 15 s and an extension step at 72°C for 160 s. Finally, the PCR was terminated by a final extension step at 72°C for 5 min. To eliminate the methylated paternal DNA plasmid, a Dpnl enzymatic digestion was conducted by adding 1 pL Dpnl (NEB) and 5 pL CutSmart buffer (NEB, finally 1x) to 44 pL PCR reaction, resulting in a final volume of 50 pL. The digestion was carried out at 37°C for 1 .5 h. The product was purified by agarose gel electrophoresis (0.8% agarose gel in 1x TAE), excision of the respective bands and applying the NucleoSpin Gel and PCR clean-up kit (Macherey-Nagel). Finally, the mutated plasmid was ligated by adding 1 pL T4 DNA Ligase (NEB) and 2 pL T4 DNA ligase reaction buffer (NEB, finally 1x) to 17 pL purified DNA. Ligation was performed at 23°C for 2 h and the enzyme was denatured at 65°C for 10 m in. The circular plasm id was transformed into electro competent E. coli BL 21 (DE3) using the same method as described for the Taq pol library preparation. After plating and incubation of the cells (37°C, overnight), 6 mL of LB medium containing 100 pg / mL carbenicillin disodium salt was inoculated with a single cell colony. Cells were grown at 37°C overnight and a glycerol stock was generated (500 pL cells mixed with 500 pL glycerol). The presence of the I707L mutation was verified by isolating and sequencing the plasmid.

[0169] Singleplexed and multiplexed one-step RT-PCR using SARS-CoV-2 RNA as template

[0170] For the singleplex PCR, the CDC-approved DNA primers and fluorescent probes that target the SARS-CoV-2_N1 , N2 and E genes were used in the reaction and are listed in Table 1. 20 pL reaction mixture contained 250 pM dNTPs (each), 670 nM forward and reverse primer (each) addressing either the nCov_N1 or nCov_N2 gene, 0.5 M betaine, 0.5 mM MgCL, 400 nM Taq pol aptamer, 120 nM respective Taq pol variant, 170 nM fluorescent probe addressing either the nCov_N1 or nCov_N2 gene and SARS-CoV-2 RNA template as indicated in 1x Taq reaction buffer (50 mM Tris- HCI (pH 9.2), 16 mM (NH^SCM, 2.5 mM MgCL, 0.1 % (v / v) Tween 20). When addressing the nCoV_E gene, the same reaction mix was used as described above, but 850 nM forward and reverse primer (each) and 216 nM fluorescent probe were applied. After preincubation at 65°C for 150 s, 10 cycles with 95°C for 5 s and 65°C for 150 s were carried out. Then, 40 cycles with 95°C for 10 s and 57°C for 35 s followed. Fluorescent intensities were measured after each of the 40 cycles. The LightCycler® 96 System (Roche) was used to monitor the reaction. To determine the detection limits, the SARS-CoV-2 RNA template was diluted as indicated and applied in the singleplexed RT-PCR assay as explained above. Cq values were plotted against the decadic logarithm of the template concentrations and linear regression was employed. PCR efficiencies (E) were calculated using the slope (m) of the calculated linear function:

[0171] E = 10(^

[0172] Linear functions, coefficient of determination and PCR efficiencies are shown in Table 4. Product formation was analysed by agarose gel electrophoresis (2.5% agarose gel in 1x TAE buffer). Reactions were performed at least twice.

[0173] Table 4: Linear function, R2values and PCR efficiencies for singleplex and multiplex RT-PCR.

[0174] Table 4: continued

[0175]

[0176] For the triplex PCR, the CDC-approved DNA primers and fluorescent probes that target the SARS-CoV-2_N1 , N2 and E genes were used in the reaction and are listed in Table 1. The same reaction volume and reaction mix was used as for the singleplex RT-PCR, but all primer / probe sets targeting the nCov_N1 , N2 and E gene were simultaneously added in the same concentrations as above. The same thermocycling protocol was conducted as for the singleplex RT-PCR above. The LightCycler® 96 System (Roche) was used to monitor the reaction. To determine the detection limits, the SARS-CoV-2 RNA template was diluted as indicated and applied in the multiplexed RT-PCR assay. Cq values for were plotted against the decadic logarithm of the template concentrations and linear regression was employed. PCR efficiencies (E) were calculated as stated above. Linear functions, coefficient of determination and PCR efficiencies are shown in Table 4. Product formation was analysed by agarose gel electrophoresis (2.5% agarose gel in 1x TAE buffer). Reactions were performed at least twice.

[0177] For the quadruplex PCR, the CDC-approved DNA primers and fluorescent probes targeting the SARS-CoV-2_N1 , N2 and E genes and the RNase P mRNA were used in the reaction (Table 1 ). The same reaction volume and reaction mix was used as for the singleplex RT-PCR, but all primer / probe sets targeting the nCov_N1 , N2 and E gene were simultaneously added in the same concentrations as before. Additionally, 670 nM forward and reverse primer (each). 170 nM fluorescent probe for the RNase P detection and 1 ng / pL universal human reference RNA were added. The same thermocycling protocol was conducted as for the singleplex RT-PCR above. The LightCycler® 96 System (Roche) was used to monitor the reaction. Furthermore, The SARS-CoV-2 RNA template was diluted as indicated and applied in the multiplexed RT-PCR assay. Cq values for were plotted against the decadic logarithm of the template concentrations and linear regression was employed. PCR efficiencies (E) were calculated as stated above. Linear functions, coefficient of determination and PCR efficiencies are shown in Table 4. Product formation was analysed by agarose gel electrophoresis (2.5% agarose gel in 1x TAE buffer). Reactions were performed at least twice.

[0178] DNA Amplicon Library preparation for NGS analysis

[0179] RT-PCR with Taq DNA polymerase variants was conducted similarly to the RT-PCR screening approach using purified enzymes. 10 pL reaction mix contained 250 pM dNTPs (each), 670 nM forward and reverse primer (each, giving the 100 bp transcript from the nCov_N1 gene, Table 1 ), 0.5 M betaine, 100 nM Taq pol aptamer, 30 nM respective Taq pol variant, 10.000 copies of SARS-CoV-2 RNA template in 1x Taq reaction buffer (50 mM Tris-HCI (pH 9.2), 16 mM (NH^SCM, 2.5 mM MgCl2, 0.1 % (v / v) Tween 20). After preincubation at 61 °C for 150 s, 10 cycles with 95°C for 5 s and 61 °C for 150 s were carried out. Then, 35 cycles with 95°C for 10 s and 57°C for 35 s followed.

[0180] To determine the DNA product concentration, each reaction mix was analysed by using the 4150 TapeStation System (Agilent), High Sensitivity D1000 ScreenTapes (Agilent) and High Sensitivity D1000 Reagents (Agilent). Sample preparation and measurement was carried out according to the guidelines of the company. Each reaction was loaded twice and 1 ng of a 100 bp reference DNA with the same sequence was applied as a control. Afterwards, remaining reaction mix was purified by agarose gel electrophoresis (2.5% agarose gel in 1x TAE buffer), excision of the respective band and applying the NucleoSpin Gel and PCR clean-up kit XS (Macherey-Nagel). Elution was achieved by adding 7 pL elution buffer to the column (for better recovery, elution was loaded again). DNA concentration was determined by using the 4150 TapeStation System (Agilent) as described above. In the next step, DNA was repaired by using 0.35 nM purified DNA, 0.1 pL PreCR Repair mix (NEB) with 100 pM of dNTP (each), 1x NAD+(provided by the manufacturer) and 1x ThermoPol reaction buffer (NEB) to reach 20 pL reaction volume. Repair was performed at 37°C for 20 min. Unique molecular identifier (UMI) introduction was obtained by applying Q5 Hot Start High-Fidelity DNA Polymerase (NEB) in a three step PCR reaction. The reaction mixture contained 20 pL repair mix, 0.02 U / pL DNA polymerase, 200 nM forward and reverse primer (each) and finally 0.2x Q5 reaction buffer to reach 25 pL reaction volume. After initial denaturation at 98°C for 2 min, two cycles with 98°C for 10 s, 62°C for 30 s and 72°C for 30 s were carried out followed by a final elongation step at 72°C for 2 min. Reaction was purified by agarose gel electrophoresis as described above. This time, buffer NTI was diluted with Milli-Q water in a ratio of 2:1 and then used for dissolving gel pieces at 50°C. DNA concentration was determined with a qPCR approach, where an DNA oligonucleotide with a similar length was applied as reference in different template concentrations (200 pM-0.01 pM). 1x NEBNext Ultra II Q5 Mastermix (NEB) was used for PCR with 1.6 pL DNA, 400 nM forward and reverse primer (each), 1x SYBR® green I in 5 pL reactionvolume. After a final denaturation step at 98°C for 1 min, a three step cycling protocol with 35 PCR cycles followed with 98°C for 10 s, 60°C for 30 s and 72°C for 30 s and a final elongation step with 72°C for 2 min was conducted. Fluorescent intensity was measured after each of the 35 cycles and melting curves were determined at the end of the reaction. For analysis, Cq values of reactions with reference DNA were plotted against the decadic logarithm of the template concentrations and linear regression was employed. DNA concentrations of samples to be analysed were calculated using the linear predictor function. Results were multiplied by two. DNA was repaired once again by using 0.05 pL PreCR mix (NEB), 1x NAD+, 100 pM of dNTP (each), 48.9 fM DNA and 1x ThermoPol reaction buffer (NEB) to reach 11.25 pL end volume. After reaction for 20 min at 37°C, DNA mix was directly used for Amplicon PCR. Here, 400 nM forward and reverse primer (equipped with Illumina adapter sequence and indices), the entire repair mix (final DNA concentration 22 fM) and 1x NEBNext Ultra II Q5 Mastermix (NEB) was used for reaction in 25 pL end volume. After a final denaturation step at 98°C for 5 min, a three step cycling protocol with 35 PCR cycles followed with 98°C for 10 s, 70°C for 30 s and 72°C for 30 s and a final elongation step at 72°C for 2 min was conducted. Reaction was analysed and purified by agarose gel electrophoresis as described above. To dissolve gel pieces, buffer NTI (Macherey Nagel) was diluted with Milli-Q water in ration 2:1 and 450 pL NTI mix was added to the gel pieces. After complete dissolving at 50°C, the mixture was diluted once again with Milli-Q water to reach a final volume of 1750 pL. DNA was loaded on the column and eluted with 20 pL elution buffer (load twice). After performing the repair reaction on the whole eluate as described above (25 pL reaction volume), DNA was purified by using the QIAEX II System (Qiagen). Here, 2 pL particle slurry was used for each sample and elution of DNA was achieved by adding 18 pL Milli-Q water to the dried beads, incubation for 5 min and taking 16 pL after centrifugation. The concentration of the final DNA library was determined by Quantus™ Fluorometer (Promega) and ranged between 19-64 nM. After quality control with Bioanalyzer 2100 (Agilent), DNA libraries were pooled and sequenced in paired-end mode (2*75 bp) on an Illumina NextSeq instrument. For RT-Taq,

[0181] Mut_RT and Taq pol V2, library preparation was performed twice. Each DNA library was sequenced once and the resulting coverage was used for error calculation (Table 5).

[0182] Table 5: Coverage for various NGS libraries generated by RT from SARS-CoV-2 RNA catalyzed by different Tag pol variants. The listed coverage values and the numbers of unique molecular identifier (UMI) families were included in the error calculation.

[0183] NGS data processing of amplicon libraries

[0184] Sequencing data were processed by using the software KNIME (Table 6). Quality and sequence are read from the FastQ files. Phred quality scores (Q scores) were transformed into base calling error probabilities (P) by using:

[0185] Readl and Read 2 were merged giving the expected size of transcript. The highest quality data was used in the overlapping regions of Readl and Read2. High quality data were filtered and aligned to the RNA reference of the SARS-CoV-2 RNA sequences. A frameshift correction was performed e.g. caused by phasing or prephasing during sequencing. Those reads with a misalignment higher than 24% were removed from the data set (removal of only 0.5% of the reads such as adapter dimers). Reads were sorted into UMI families and family read numbers were counted. Substitution error calculation was performed in each UMI family having more than 3 reads. If 90% of the reads within each family carry the misincorporation, the error is set to 1 (otherwise 0). Afterwards, the substitution error was calculated over all UMI families at each position of the transcript. To normalize the error rates of each Taq pol variant according to its amplification activity, the individual amplification and doubling numbers of each enzyme were included in the calculation. The amplification number (A) and the doubling number (D) were determined by using the following equations (n=amount of substance [mol]):

[0186] Each error rate for each position in the template was divided by the individual doubling number and plotted using Microsoft excel software. For the average substitution error, the region of the amplicon without primer binding sites is included (area depicted in Fig. 11 ). The last T read in this region was excluded since values on this positions differ very clearly from the rest of the data. The coverage, the number of UMI families and the doubling numbers used for error calculation are listed individually for each sequencing library in Table 5. Table 6: Software used in this study.

[0187] Example 1 :

[0188] Screening for Tag DNA polymerase variants with improved RT-PCR activity

[0189] Previous studies have identified the RT-active KTq DNA pol (RT-KTq) carrying 4 amino acid mutations namely L459M, S515R, I638F and M747K (Fig. 1 A and C, amino acid side chains depicted in blue). The enzyme was obtained after a recombined DNA shuffling approach that combined seven distinct mutations previously reported to promote either reverse transcription or to expand the substrate spectrum. After pre-selection for both PCR- and RT-activity, the RT-KTq showed up to 100-fold increased RT compared to the applied parental enzymes. In addition, the previous combination of a rational and a combinatorial enzyme library design revealed Mut_RT DNA pol carrying five mutations, namely N483K, E507K, K540Y, V586G and I614K (Fig. 1 A and C, amino acid side chains depicted in green). This enzyme exhibited a significantly increased activity on both RNA and DNA in comparison to the parental enzyme.

[0190] Generally, RT-KTq and Mut_RT were evolved from two enzyme libraries that were constructed independently from each other by different approaches. As consequence, two different pools of mutations were reported to successfully increase RT activity. It was reasoned that recombination of proficient mutations stemming from both enzymes might result in even more empowered DNA pol with RT activity. In addition, since the full length Taq pol was shown to be more processive than its truncated version and the insertion of RT-boosting mutations into the Taq pol backbone further increased the RT-activity compared to its respective KTq pol variant, this scaffold was used. Thus, the 4 mutations of the RT-KTq were introduced in the Taq pol backbone to form the RT-Taq (Fig. 1 B and C bottom, amino acid side chains depicted in blue). In preliminary experiments with the RT- KTq and all 5 single mutants of the Mut_RT, it was identified that not all mutation sites contribute equally to a better enzyme performance. KTq K540Y showed neither activity in primer extension nor in the RT-PCR experiments (Fig. 2). As consequence, this mutation was not included in the following screening approach. The experiment demonstrates however that the combination of different RT-boosting mutations in Mut_RT resulted in positive synergistic effects, which together improve the enzyme performance.

[0191] To recombine the differently evolved RT enhancing mutation pools, a new enzyme library of Taq pol mutants was designed including all possible combinations of L459M, N483K, E507K, S515R, V586G, 1614K, I638F and M747K with the respective wild-type amino acids. Thus, the library comprised the wild-type Taq pol without mutations, as well as all single, double, triple and 4-8-fold mutants, resulting in 256 Taq pol variants in total (Fig. 1 D). In this way, the RT-Taq and the Taq pol variant bearing the Mut_RT specific mutations were included, as well. The genes of all polymerase variants were synthesised in equimolar amounts, inserted into the pGDR1 1 vector, and used as a mixture. The plasmid mix was transformed in E. coli BL21 (DE3) cells and plated on agar plates. A total of 2660 single colonies were picked and arranged in a 348-well format. Oversampling by a factor of more than 10 during colony picking ensured that more than 99% of all possible Taq pol variants were covered and included in the library. Cell lysates from expression cultures were directly used after heat inactivation of E. coli host proteins. The screen comprised a real-time RT-PCR approach that was previously established to detect the SARS- CoV-2 RNA in unprocessed swap samples.

[0192] Primers and TaqMan probes used in the study are similar to those applied in the FDA emergency-use authorized CDC (Centers for Disease Control and Prevention) diagnostic assay and target the nucleocapsid gene N1. After the precise and efficient PCR, a 72 bp DNA amplicon is generated. The SARS-CoV-2 RNA was ordered from the European Commission Joint Research Centre (CRM code: EURM- 019) and included in vitro transcribed single-stranded RNA from the SARS-CoV-2. The formation of the cDNA can be followed by monitoring the increase in fluorescence signal since the hydrolysis probe is digested by the Taq pol during the ongoing PCR, leaving the unquenched FAM (6-carboxyfluorescein) dye in the reaction mix (Fig. 3 A).

[0193] The reaction conditions were optimised for the use of expression lysates and the RNA template concentration of 1000 c / pL (copies / pL) was found to be sufficient to adequately monitor the enzyme performance (Fig. 4). After screening 2660 cell lysates, promising Taq pol variants were selected based on lower Cq values than those of the RT-Taq and amplification curves following a sigmoidal shape (Fig. 3 B). The best 379 variants were selected and corresponding cells were transferred to a new 384 deep well plate. 5 positions on the plate were occupied by E. coli cells carrying the RT-Taq vector. The cell lysates were screened again by applying a RNA template dilution from 1000-1 c / pL and different forward primer to generate PCR products with increasing lengths (72, 100, 156, 208 and 254 bp). Finally, 20 Taq pol variants showed good performance in all reaction conditions (Fig. 5) and 46 variants performed well in only 8 from 9 conditions. The expression levels were checked for uniformity by SDS-PAGE (Fig. 6 A). In order to determine the mutation patterns of the 20 most promising Taq pol variants, plasmids from corresponding E. coli cells were isolated and sent for Sanger sequencing. 15 different DNA genes were found since some E. coli cells carried the same plasmid encoding for the same Taq pol variant (Table 7).

[0194] Table 7: Composition of the mutation sites found in the 20 most promising Taq pol variants. Corresponding cell lysates are indicated.

[0195] Mutation sites originate from RT-KTq are coloured in blue (mutation sites 1 , 4, 7, and 8) and mutation sites originate from Mut_RT are coloured in green (mutations sites 2, 3, 5, and 6). Positions with mutations are highlighted in bold letters.

[0196] Moreover, some plasmids from E. coli cells whose lysates showed no activity were also analysed by Sanger sequencing (Table 8). To ensure that lower activity was not due to low expression levels, only those lysates were selected that show protein expression upon SDS-PAGE analysis (Fig. 6 B).

[0197] When comparing the mutation composition of active and inactive Taq pol variants, it is striking that the mutation I638F only occurs in the inactive variants (Fig. 3 C). 1614K was the most common mutation in the active variants, while it was less present in the inactive ones. Interestingly, most of the well performing variants carried only 3 mutations in their amino acid sequence (Fig. 3 D, left). However, within the group of inactive variants, there were some enzymes with a higher mutation load (Fig. 3 D, right). These two aspects indicate that not only the composition of RT- promoting mutations but also a moderate mutation load of approximately 3 mutations contributed to a well performing enzyme.

[0198] Table 8: Composition of the mutation sites found in inactive Taq pol variants. Mutation sites originate from RT-KTq are coloured in blue (mutation sites 1 , 4, 7, and 8) and mutation sites originate from Mut_RT are coloured in green (mutations sites 2, 3, 5, and 6). Positions with mutations are highlighted in bold letters

[0199]

[0200] Further characterization of variants

[0201] For further studies, all 15 promising RT-active Taq pol variants were purified and subjected to a similar screen as described above for the study on the most promising E. coli cell lysates. For this purpose, a RNA template dilution from 1000-1 c / pL was used to further increase the stringency of the assay conditions. Furthermore, RT- PCR was performed with different forward primers to generate PCR products of increasing length (72, 100, 156, 208 and 254 bp). Of note, the same reaction conditions were selected for all approaches without any further optimisation, even if there was low input of RNA or longer amplicons had to be generated. Taq pol V2, V3, V7, V9, V11 , V12, V13 and V15 showed the best amplification curves over all conditions (Fig. 7). Product formation was observed even in more demanding reactions, such as those with very low RNA template input or the synthesis of longer amplicons. Similar to reactions performed with E. coli lysates, RT-Taq was not able to amplify from RNA templates with low concentrations and to form longer PCR products (Fig. 7, red curves).

[0202] Next it was investigated if the identified Taq pol variants tolerate SYBR® green I in real-time PCR. Therefore, all 15 Taq pol variants were applied again in the RT-PCR assay using SARS-CoV-2 RNA with different combinations of reverse and forward primers. Similar to observations above Taq pol V2, V3, V7, V9, V11 , V12, V13 and V15 showed the best amplification curves over all reactions (Fig. 8 A). Furthermore, the experiment showed that the improved RT-PCR activity of the identified Taq pol variants is not only due to a higher nuclease activity, but most importantly due to an improved PCR activity.

[0203] In order to examine whether the purified Taq pol variants can catalyse RT-PCR without a prior RT step, we performed amplification from an RNA oligonucleotide under time-limited conditions. Here, the cDNA was obtained by direct heating to 95°C and subsequent conducting a two-step amplification. For comparison, the RT- KTq, Mut_RT and RT-Taq and all 15 Taq pol variants were tested using the same reaction parameters as shown in Figure 2. The amplification curves measured for Taq V2, V3, V7, V9, V12 and V15 passed the threshold at the same time or earlier than RT-KTq, Mut_RT or RT-Taq (Figure 8 B). The highest activity was observed for Taq pol V2 and V3 starting the DNA amplification after a low number of cycles and generating a high fluorescence intensity. Indeed, this experiment demonstrated that the most effective Taq pol variants identified in the screening did not need an extra RT step prior to amplification. Considering all RT-PCR experiments with purified enzyme, Taq pol V2, V3, V12 and V15 showed the best performance in terms of low Cq values and amplification curves with high fluorescence intensities. RT-PCR from the SARS-CoV-2

[0204] RNA

[0205] Having established that Taq pol V2, V3, V12, V15 can faithfully catalyse RT-PCR from the SARS-CoV2 RNA targeting the nCov_N1 gene, it was investigated whether other targets could be detected using this approach. RT-PCR was performed using the primer and probe sets for the nCov_N2 and nCov_E genes that correspond to the FDA-approved CDC emergency diagnostic assay. Initially, all gene targets were monitored individually in order to find a suitable reaction condition for all of them. Moreover, the additional mutation I707L was introduced into the backbone of the Taq pol V2 (henceforth referred to as Taq pol V2 IL) to investigate whether this can further improve PCR activity. It had already been shown that I707L reduces the polymerase activity at lower temperatures, while retaining normal activity at the optimal reaction temperature of the KTq pol enzyme. Taq pol V2 was selected as an example because of its excellent performance in previous experiments.

[0206] All variants of the Taq pol were able to catalyse the reaction from each SARS-CoV- 2 gene under the same optimised reaction condition (Fig. 9 A). Taq pol V15 is partially excluded here, as it did not show an increase in the fluorescence signal for the Texas Red dye and thus detection of the nCov_E gene was not successful. The parental enzyme RT-Taq was only able to amplify products from the nCov_N2 gene and to a much lesser extent from the nCov_N1 gene (Fig. 9 A, red curves). Of note, detecting the nCov_E gene is more challenging than detecting the other targets because the other amplicons are shorter in length (72 bp for nCov_N1 , 67 bp for nCov_N2 and 113 bp for nCov_E). This experiment benefits from the exceptional thermostability of the enzymes since RT was carried out at 65°C.

[0207] Next, the Taq pol variants were challenged by gradually reducing the template concentration to determine the limit of detection. With this reaction setup, it should be noted that uniform reaction conditions were defined for all genes to allow simultaneous detection. Consequently, the conditions for nCoV_E were not individually optimized. Since the Taq pol V2 IL had previously shown an improved performance on the nCov_E gene compared to the Taq pol V2, this variant was considered further. The Cq values were plotted against the decadic logarithm of the template concentration and linear regression was applied (Fig. 9 C, Table 4). The parental RT-Taq pol was able to detect 100 copies of the SARS-CoV-2 template (5 c / pL) by monitoring the nCov_N2 gene. The dilution series was amplified with a PCR efficiency of 91 %. Other targets could not be detected adequately (Fig. 9 C, left). Taq pol V2, V3 and V2 IL showed the lowest detection limits with 20, 10 and 20 copies respectively (Fig. 9 C, three plots right). All dilution series had a PCR efficiency above 90% except for the reaction from the nCov_E gene. These results render Taq pol V2, V3 and V2 IL as promising candidates.

[0208] In the next step, it was investigated if the nCov_N1 , nCov_N2 and the nCov_E gene could be monitored simultaneously in one reaction. The promising Taq pol variants V2, V3, V12, V15 and V2 IL were applied in multiplexed RT-PCR under similar conditions as in the singleplex reaction setup (Fig. 9 B). At a template concentration of 10.000 c / pL, all DNA pols, except Taq pol V15 and the parental RT-Taq, successfully amplified all three genes. As in the previous experiments, the concentration of the SARS-CoV-2 template was gradually decreased, the corresponding Cq values were plotted against the decadic logarithm of the template concentration and linear regression was applied (Figure 9 D, Table 4). The sensitivity of the parental RT-Taq pol was reduced from 100 (singleplex) to 2000 copies by monitoring the nCov_N2 gene. PCR efficiency was determined to be 104%. However, Taq pol V2 and V2 IL were still able to detect 100 copies, while Taq pol V3 identified only 20 copies. Thus, the latter is 100 times more sensitive than the parental RT-Taq pol. The PCR efficiencies are consistently above 92%, with the exception of the reactions from the nCov_E gene (Table 4). Overall, this demonstrates that Taq pol V2, V3 and V2 IL are able to catalyse RT-PCR in singleplex and multiplex reaction formulations and independently of the RNA target sequence.

[0209] It was then tested whether the amplification from the human RNase P transcript could be used as an internal control reaction to verify the integrity of the patient samples in the future. For this purpose, RT-Taq, Taq pol V2, V3 and V2 IL were used in multiplexed RT-PCR targeting the SARS-CoV-2 nCov_N1 , nCov_N2, nCov_E gene and the RNase P transcript simultaneously (quadruplex RT-PCR). The universal human reference RNA (Thermo Fisher Scientific) was used as template and the primer / probe sets from the FDA authorized CDC diagnostic assay were applied for catalysis. At the template concentrations of 10.000 c / pL of SARS- CoV-2 and 1 ng / pL human reference RNA, all DNA pols were able to successfully amplify the 4 target sequences (Fig. 10 A). However, the parental RT-Taq pol was only able to catalyse the reaction from the nCov_N2 gene and the RNase P transcript (Fig. 10 A, red curves). Of note, the reaction conditions for the quadruplex reaction did not need to be optimised again.

[0210] To further investigate the enzyme performance, the SARS-CoV-2 RNA was diluted to 500 c / pL in 4 steps, while keeping the human reference RNA constant at 1 ng / pL. The respective Cq values were plotted against the decadic logarithm of the template concentration and linear regression was applied (Fig. 10 B, Table 4). The best performance was observed for Taq pol V3, as all 4 RNA targets were successfully monitored at each dilution step and PCR efficiencies ranged from 93% to 104%. Furthermore, the Cq values for reactions from the RNase P transcript remained constant at approximately 13.9. The successful detection of SARS-CoV-2 observed in the triplex RT-PCR was therefore also achieved in the presence of a different RNA target, demonstrating the potential applicability of an internal standard in the reaction.

[0211] Taken together, it was demonstrated that the newly developed Taq pol variants can be successfully used in single- tube RT-PCR approaches without the need for a second enzyme. As the human reference RNA used in this study is a mixture of RNA extracts from 10 cell lines, SARS-CoV-2 was successfully detected from a complex mixture of RNA. Moreover, the present data shows for the first time an example of a one-step quaduplex RT-PCR using only one single DNA polymerase.

[0212] Finally, the polymerase fidelity was investigated within the RT-PCR process. Therefore, the parental enzymes RT-Taq, Mut_RT, Taq pol V2, V3 and V2 IL were used to reverse transcribe and amplify a 100 nt segment of the nCov_N1 gene and the resulting amplicons were subsequently subjected to NGS (Fig. 11 ). RT-Taq and Mut_RT showed comparable error rates and revealed an average error of 0.07%. Taq pol V2, V3 and V2 showed slightly increased error patterns compared to the parental enzymes resulting in average errors of 0.21 %, 0.19%, and 0.14%, respectively. It should be noted that the KTq pol generally has a higher accuracy than the full-length Taq pol. The introduction of the I707L mutation into the Taq V2 pol variant increased the polymerase fidelity by a factor of 1.5. Of note, naturally occurring RTs are known to have lower fidelity than DNA-dependent DNA polymerases. Therefore, the catalysis during RT-PCR is likely to be more error prone. The Taq pol variants used in this study are DNA polymerases capable of accepting and processing RNA while successfully amplifying DNA. Thus, errors might not be primarily introduced in the first linear RT step, but also during amplification. Inspecting the error distribution for each nucleobase identity separately, shows that all DNA polymerases exhibit the highest error rates for U, followed by A (Fig. 12). The highest fidelity was observed for the incorporation of nucleotides opposite G and C bases. erase variants

[0213] In order to compare RT-Taq and RT-KTq variants regarding linear RT- and PCR activity, the 4 mutations of the RT-KTq (namely L459M, S515R, I638F and M747K) were introduced in the Taq pol backbone to form the RT-Taq. To investigate the enzyme performance in terms of RT and PCR activity, the RT-Taq enzyme as well as the RT-KTq and Mut_RT as reference, were applied in multiple incorporation primer extension experiments with radioactively labelled primers and RT-PCR experiments (Figure 13). The RNA oligonucleotide was used as template. Both RT- Taq and Mut_RT extended more primer to the full-length product, indicating a higher linear RT activity compared to the RT-KTq (Figure 13 A). In RT-PCR, cDNA amplification started after 9 (RT-KTq), 10 (RT-Taq) and 11 cycles (Mut_RT), but the highest fluorescent intensity was measured for the amplification catalyzed by Mut_RT (Figure 13 B). The analysis of the product formation by agarose gel electrophoresis showed that both RT-Taq and Mut_RT generated more cDNA than RT-KTq (Figure 13 C).

[0214] These experimental results show that, with respect to the mutation patterns described herein, the use of the complete Taq enzyme scaffold, in contrast to the truncated KTq variant, has a positive influence on enzyme activity.

[0215] Discussion (Examples 1 to 4):

[0216] The present invention reports on the development of RT-PCR active DNA polymerases, Taq pol V2, V3 and V2 IL, that differ by only 3-4 amino acids from wild-type Taq pol. The enzymes are capable of catalysing one-step RT-PCR. Moreover, these DNA pols are also capable of performing multiplex RT-PCR and thereby represent the first reported enzymes that are capable of performing multiplex RT-PCR with a single enzyme. Their use in well-established RNA detection approaches can not only improve current laboratory procedures in terms of time and cost, but also offers the possibility of addressing complex RNA targets due to their high thermostability. The Taq pol variants were discovered in a screening approach that combined two independently discovered pools of mutations enhancing the RT activity of the parental KTq pol. The full length Taq pol was used as library scaffold because it has been shown to be more processive than its truncated version. Moreover, the Taq pol scaffold allows their employment in hydrolysis probe-based test systems that are widely used in molecular diagnostics. Several DNA pol variants were identified that are capable of detecting the exemplary chosen SARS-CoV-2 genome in the N1 region, even when the RNA template was diluted and the enzyme had to generate longer DNA amplicons. Further analysis and characterisation lead to the identification of Taq pol V2, V3 and V2 IL carrying the mutations N483K, E507K, 1614K (Taq pol V2), N483K, S515R, 1614K (Taq pol V3) and N483K, E507K, 1614K, I707L (Taq pol V2 IL). These enzymes were able to detect the N1 , N2 and E gene of SARS-CoV-2, both individually and simultaneously with sensitivity of down to 10 (singleplex RT-PCR) and 20 (multiplex RT-PCR) copies of RNA. Application in a quadruplex RT-PCR assay demonstrated simultaneous monitoring of all three SARS-CoV-2 genes from a complex mixture of RNA and successful detection of the RNase P transcript to ensure the integrity of the patient sample.

[0217] The one-enzyme formulation has several advantages over a two-enzyme based RT- PCR. Reaction parameters can be optimally adapted to a single DNA pol variant. This means that there is no need to find a compromise between the reaction conditions of two enzymes. Furthermore, the thermostability and the usage of the Taq pol aptamer enable a hot-start of the reaction minimizing the propensity for false amplification in case of high structured RNA. Besides, RT-PCR does not necessarily require an extra RT step prior to amplification, which saves time. Moreover, there is no competition for the binding and thereby blocking of the RNA template, as may occur with two enzymes, which could potentially improve efficiency of detecting small amounts of the target molecules. Since only 3 or 4 mutations need to be introduced into the wild-type Taq pol backbone, established production and purification processes can simply be retained, which saves current resources. The mechanism by which these polymerases acquire their properties is currently investigated. All mutation sites are located within the polymerase domain, which likely allows for the proper binding of the DNA / RNA hybrid and / or the folding of the polymerase into its active form after DNA / RNA binding. It is known that amino acid N483 is located in the thumb domain of the Taq pol contacting the template strand. Residues E507 and S515 are also part of the thumb domain and interact with the primer strand. Especially, the mutation E507K has been shown to contribute to a fast PCR cycling property of the Taq pol. The amino acid 1614 is located in the finger domain and forms part of the hydrophobic pocket that binds the nucleobase and deoxyribose of the incoming dNTP. Its mutation has been identified to affect the fidelity of nucleobase pairing. Mutation I707L is placed in the finger domain and has been shown to reduce the polymerase activity at lower temperatures, while retaining normal activity at the optimal reaction temperature and thermostability at 95°C. Overall, the mutation sites are distributed throughout the polymerase domain and might contribute to synergistic effects that are improving the properties of RT-PCR. The present invention adds new DNA polymerases to the toolbox of enzymes suitable to be used in RT-PCR methodologies and further demonstrate that DNA polymerase engineering is a viable strategy to obtain enzymes with suitable properties.

[0218] Example 5:

[0219] Fifteen mutant Taq polymerases that have been previously identified in a screening were evaluated for enzymes with elevated RT activity. The SARS CoV2 target N1 was amplified from 10.000 copies of an in vitro transcript in a one step RT PCR reaction, resulting in a 72bp amplicon. Amplification was performed on a BioRad CFX 384 instrument using cycling conditions as outlined in Table 9. As a reference, the QuantiNova Multiplex RT-PCR Kit (QIAGEN, Hilden) was used according to the manufacturer’s protocols in a separate run. Data was analysed using the BioRad Maestro software. Variants with the mutations N483K E507K 1614K, or N483K S515R 1614K were found to show the best performance when used as sole enzyme in a one-step RT PCR. Surprisingly however, when comparing all variants in the presence of a wildtype Taq polymerase, another variant showed superior performance, indicating that it gained the best RT activity, but significantly lost DNA polymerase activity (Fig. 14). The Taq mutant described here carries the amino acid substitutions N483K, S515R, 1614K, and M747K with regard to wildtype Taq polymerase (SEQ ID NO 1 ). This mutant is referred to as “mutant V6” herein. Note that data collection was done during the final step only so that the first 10 cycles of the previous step are not covered in Ct values shown in Fig. 14.

[0220] Amplification plots illustrate that mutant V6 does not show exponential DNA amplification that lead to significant levels of product (Fig. 15).

[0221] Example 6:

[0222] In another experiment, the sensitivity of mutant V6 was compared to mutant V3 in more complex assays (Fig. 16). The two enzymes only differ in one amino acid, with V6 having the amino acid substitutions M747K in otherwise identical background. Here, total human RNA (Human Xpress Ref Universal Total RNA, QIAGEN) was used in different amounts as template. Two probe-based assays were performed targeting the higher abundant ACTB (beta-actin) and the lower abundant p68 transcript. As in the previous experiment, wildtype Taq polymerase was present along with the mutant enzymes. Each Taq enzyme was used with 2.5U per reaction in the QuantiNova Multiplex RT PCR reaction buffer. The PCR was performed on a BioRad CFX 384 instrument using cycling conditions as outlined in Table 9. Consistently lower Ct values (app. 2 Ct difference) were observed with mutant V6 with both targets.

[0223] Next, it was investigated if cDNA synthesis can be separated from DNA amplification as in traditional two step RT-PCR protocols depending on reaction conditions. Furthermore, it was attempted to find out if the mutant enzyme (V6) allows to create multiple copies of individual RNA molecules by running cycles of denaturation and annealing / extension phases. The SARS CoV2 N1 assay was used with Taq variant V6. 1 , 5, or 10 cycles with identical overall reaction time (1 x 1500 sec, 5 x 300 sec, 10 x 150 sec, with conditions of Table 9) were run in the absence of a wildtype Taq enzyme, and with either only the primer complementary to the RNA strand, or both primers present. Reactions were complemented with 2.5LI wildtype Taq and primer afterwards for the remaining amplification. The PCR was performed on a BioRad CFX 384 instrument and using the QuantiNova Multiplex PCR (QIAGEN, Hilden) reaction mix. Results indicate that RNA is efficiently reverse transcribed multiple times, as concluded from Ct shifts approximately reflecting linear amplification with only one primer present (Fig. 17). This also shows that the mutant enzyme V6 maintains RT activity along at least 10 rounds of denaturation at 95°C. With both primers present, final Cts are lowered by the number of cycles ran, indicating that exponential amplification happened under these conditions. Note that this also means that Taq V6 alone is efficiently amplifying DNA under these conditions, although it does not seem to be able to amplify to significant final product amounts without the help of a wildtype Taq.

[0224] To further show the difference in heat stability of the mutant Taq V6 in comparison to a regular RT enzyme, the cycling protocol of Table 9 was used on a QIAcuity dPCR instrument to detect the ACTB gene and an internal control (QuantiNova IC RNA, QIAGEN). Reactions were carried out in the QIAcuity OneStep Advanced master mix with 2.5LI of mutant Taq V6, the kit furthermore was used as a reference including the OneStep Advanced RT mix. The mutant Taq detected ACTB in copy numbers correlated to the input RNA amount and a stable IC. In contrast only background noise is seen with the RT reference reactions (Fig. 18).

[0225] Table 9: Cycling conditions.

[0226] Conventional RT enzymes are known to inhibit Taq polymerase activity if in excess, so that increasing RT activity in most cases requires to also add more Taq. It was explored if such inhibition as well occurs with the mutant Taq variants, in particular for V6, as it has poor DNA polymerase activity, but may still bind the template. V3 and V6 were mixed with regular Taq (always at 2.5LI) in ratios 1 :1 up to 1 :10 in QuantiNova Multiplex RT-PCR master mix. 1 ng RNA (XpressRef Universal Total RNA, QIAGEN) was used as a template with the ACTB150 assay. The PCR was performed on a BioRad CFX 384 instrument using cycling conditions as outlined in Table 9. No significant impact of increased amounts of RT Taq was observed (Fig. 19).

[0227] Example 10:

[0228] Since Taq polymerases typically are more inhibitor tolerant than RT enzymes, robustness was tested in digital PCR (dPCR) on the QIAcuity instrument (cycling conditions according to Table 9) using hematin as a model. Two setups were used: 4U RT Taq V6 + 12U Taq, and 12U RT Taq + 12U Taq in QIAcuity HighMultiplex Probe PCR reaction mix with 20 - 200pM hematin (Sigma Aldrich) final concentration in the assay. The ACTB assay and the internal control (QuantiNova IC RNA) were monitored using 1 ng RNA (XpressRef Universal Total RNA, QIAGEN)

[0229] Stable ACTB and internal control copy numbers were observed up to the highest concentration with 12U RT Taq, with 4U a slight drop was observed at 150pM, values further decreasing to app. 10% at 200pM (Fig. 20). Scatter plots did not show increased rain for these samples, indicating that the inhibition in this case is clearly on RT activity, as also seen with conventional RTs, but sets in at much higher concentrations.

[0230] QIAcuity OneStep Advanced Probe Mix (QIAGEN) was tested as a reference according to the manufacturers recommendations and almost complete inhibition was observed already at the lowest concentration of 20pM hematin (Fig. 21 ). Enzyme and BSA concentrations of this reference mix are equivalent to the 4U RT Taq setup, so that the difference can be attributed to the nature of the enzyme.

[0231] 11 :

[0232] Reverse transcriptases are able to create dimers from oligonucleotides with complementary 3’ ends which can cause issues in assays without probes (e.g. SYBR Green), or in multiplex reactions that include many different oligonucleotides. Warm start mechanisms for RT enzymes have been established, e.g. using aptamers. Some mechanisms to reduce residual activity at room temperature established for Taq polymerases however are not applicable to RT enzymes, because of the high temperature required for activation, for example antibodies and hot start dNTPs. In order to test if both, low and high temperature release of inhibition options are applicable for the mutant Taq polymerase V6, two oligos were incubated with 4 complementary 3’ bases in the presence or absence of Taq aptamer (SEQ ID NO: 6), Taq antibody (QIAGEN), and hot start dNTPs (TriLink CleanAmp dNTP Set). Incubation was done for 17 hours at ambient temperature. After incubation, a wildtype Taq was added to amplify primer dimers that had formed in a probe based real time PCR on a QuantStudio 5 instrument (Applied Biosystems). The addition of aptamer, antibody, and modified dNTPs all led to increased Ct values, indicating less primer dimers have been formed. Combining the individual components led to further reduction (Fig. 22).

[0233] Discussion (Examples 5 to 11 ):

[0234] These results of the above examples allow the following conclusions:

[0235] The DNA polymerase V6 according to the present invention acts as a highly heat stable reverse transcriptase (RT), is highly inhibitor tolerant, can be inactivated by hot start mechanisms established for Taq polymerases, can be used like a traditional RT in two-step RT, but with the option to fully denature RNA and to run cDNA synthesis at elevated temperatures to facilitate conversion of difficult targets, can do multiple cDNA conversions from individual RNAs, e.g. to enrich cDNAs in a two-step reaction, and can do RT and DNA amplification at the same time and thereby allows fast protocols.

Claims

62Claims1. A DNA polymerase derived from wild-type Thermus aquaticus (Taq) DNA polymerase, comprising the mutations N483K, S515R, 1614K, and M747Kwith regard to the amino acid sequence of wild-type Taq DNA polymerase (SEQ ID NO: 1 ).

2. A DNA polymerase derived from wild-type Thermus aquaticus (Taq) DNA polymerase, comprising(i) the mutations N483K, E507K, and 1614K;(ii) the mutations N483K, S515R, and 1614K;(iii) the mutations E507K, and 1614K;(iv) the mutations L459M, E507K, and 1614K;(v) the mutations N483K, E507K, S515R, and 1614K;(vi) the mutations L459M, N483K, S515R, and 1614K;(vii) the mutations L459M, N483K, E507K, V586G, and 1614K; or(viii) the mutations N483K, E507K, S515R, V586G, and 1614K; with regard to the amino acid sequence of wild-type Taq DNA polymerase (SEQ ID NO: 1 ).

3. The DNA polymerase of claim 2, comprising one of the mutation patterns (i) to (iv) with regard to SEQ ID NO: 1 .

4. The DNA polymerase of claim 3, comprising mutation pattern (i) or (ii) with regard to SEQ ID NO: 1 .

5. The DNA polymerase of any one of claims 2 to 4, further comprising the mutation I707L with regard to SEQ ID NO: 1 .

6. The DNA polymerase of any one of claims 1 to 5, comprising the amino acid sequence as shown in SEQ ID NO: 1 including said mutations.

637. The DNA polymerase of any one of claims 1 to 5, comprising the amino acid sequence corresponding to(i) amino acids 293 to 832 of SEQ ID NO: 1 including said mutations,(ii) amino acids 4 to 832 of SEQ ID NO: 1 including said mutations,(iii) amino acids 279 to 832 of SEQ ID NO: 1 including said mutations, or(iv) amino acids 290 to 832 of SEQ ID NO: 1 including said mutations.

8. The DNA polymerase of any one of claims 1 to 7, wherein said DNA polymerase does not comprise any further mutations with regard to SEQ ID NO: 1.

9. A nucleic acid comprising a nucleotide sequence coding for a DNA polymerase according to any one of claim 1 to 8.

10. A vector comprising the nucleic acid of claim 9.

11. A host cell comprising the vector of claim 10 or the nucleic acid of claim 9.

12. A reverse transcription polymerase chain reaction (RT-PCR) method, comprising the steps:(a) reverse transcription of one or more RNA(s) of interest to cDNA, and(b) amplification of the cDNA generated in step (a) by PCR, wherein step (a) is catalyzed using a DNA polymerase of claim 1 , or of any one of claims 6 to 8 as far as dependent on claim 1 .

13. The method of claim 12, wherein step (b) is catalyzed by a second DNA polymerase.

14. The method of claim 13, wherein the second DNA polymerase is wild-type Taq DNA polymerase.6415. The method of claim 12, wherein both of steps (a) and (b) are catalyzed using a DNA polymerase of claim 1 , or of any one of claims 6 to 8 as far as dependent on claim 1 .

16. A reverse transcription polymerase chain reaction (RT-PCR) method, comprising the steps:(a) reverse transcription of one or more RNA(s) of interest to cDNA, and(b) amplification of the cDNA generated in step (a) by PCR, wherein both of steps (a) and (b) are catalyzed using a DNA polymerase of any one of claims 2 to 5, or of any one of claims 6 to 8 as far as dependent on claim 2.

17. The method of any one of claims 12 to 16, wherein both of steps (a) and (b) are performed concurrently and in the same reaction vessel.

18. The method of any one of claims 15 to 17, wherein no other enzyme using a nucleic acid as substrate is present during steps (a) and (b).

19. The method of any one of claims 12 to 18, wherein more than one RNA of interest is reversely transcribed in step (a).

20. The method of any one of claims 12 to 19, wherein the PCR is a quantitative PCR (qPCR) or digital PCR (dPCR).21 . A kit comprising the DNA-polymerase of any one of claims 1 to 8.

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