Method for improving nucleic acid processing

Abstract

The present invention relates to a combination of agents for improving nucleic acid processing, such as amplification or sequencing, a method for improving nucleic acid processing involving such combination of agents, a composition for use in such a method, and to a kit associated with such method.

Description

Method for improving nucleic acid processing

[0001] The present invention relates to a combination of agents for improving nucleic acid processing, such as amplification or sequencing, a method for improving nucleic acid processing involving such combination of agents, a composition for use in such a method, and to a kit associated with such method.FIELD OF THE INVENTION

[0002] The present invention relates to the field of molecular biology, more particularly to the processing of nucleic acid molecules which tend to form secondary structures and / or have high GO content.BACKGROUND OF THE INVENTION

[0003] Accurate and precise nucleic acid amplification and quantification via, e.g., PCR is crucial for several applications and reasons. One important application is the analysis of gene expression. When studying the expression of specific genes or comparing the expression levels among different samples or experimental conditions, accurate and precise quantification allows to obtain data which can be reliably interpreted. This holds also true for the detection of rare nucleic acid targets enabling early disease diagnosis, monitoring and evaluation of treatment response. PCR-based quantification of nucleic acids is also used in the field of drug development and pharmacokinetics. Optimal dosing is highly dependent on an accurate and precise quantification. Digital PCR (dPCR) allows for an absolute quantification without the need of standard curves or reference standards which makes the quantification even more reliable compared to a traditional quantitative PCR (qPCR).

[0004] The performance of a PCR, whether qPCR or dPCR, is dependent on several factors: 1. Assay design. The design of primers and probes is crucial for the PCR performance. Well-designed assays, melting temperature and target specificity is key. 2. PCR cycling conditions. The annealing / extension temperature as well as extension timeand number of cycles can affect PCR performance and with that accuracy and precision. 3. Template containing the target. The quality of the template also plays an important role. High loads of contaminants or low-quality DNA / RNA might negatively affect PCR efficiency. Another important aspect is the accessibility of the nucleic acid target of interest for the PCR machinery. High GC content targets or targets in a high GC-content neighborhood as well as nucleic acid targets with highly stable secondary structures such as the inverted terminal repeats (ITRs) of adeno-associated virus (AAV) genomes or long terminal repeats (LTRs) of lentivirus genomes might be difficult to melt and linearize, negatively affecting PCR performance.

[0005] In a nutshell, all above mentioned factors are important starting points for PCR optimization. However, all steps are closely related to each other and must be evaluated together.

[0006] This holds true for all types of PCR, RT (reverse transcription) PCR as well as RT reactions. Challenges provoked by, e.g., secondary structures or high GC content sequences, might be faced in an endpoint PCR / RT-PCR, qPCR / RT-PCR, digital PCR / RT-PCR as well as in a two-step reverse transcription reactions setup.

[0007] Amplification of nucleic acid targets via PCR (e.g., qPCR, dPCR) is for most applications relatively easy and robust and usually requires little adjustments for optimal performance. However, there are instances in which a particular nucleic acid target region is difficult to amplify due to, for example, high GC content or stable secondary structures. GC-rich sequences are known to be challenging to amplify due to the formation of secondary structures and G-quadruplexes. The ITRs of adeno-associated viruses (AAVs) are around 145-160 bp long and have palindromic sequences that fold in a t-shaped structure. They are crucial elements that facilitate for example viral replication and packaging. However, for analytical procedures, those structures have a rather challenging impact. Their high GC content and stable secondary structures make amplification and sequencing very difficult. Those structures can prevent for example primers and / or probes to bind to the dedicated region or prevent progression of the DNA polymerase. In those cases, the quantification may result lower than expected underestimating the "real" titer or in unspecific PCR products.

[0008] In an attempt to overcome challenges in amplification, the assay design (e.g., sequence, melting temperature, etc.) is adjusted. Additionally, the magnesium concentration, the pH of the buffer, PCR cycling and number of cycles can be optimized with no guarantee for success. Alternatively, a variety of additives and enhancing agents can be used to increase yield, specificity and consistency. Supplements such as DMSO, formamide, glycerol, detergents, BSA, PEG, TMA and betaine can have beneficial effects on some PCR amplifications. Nevertheless, it cannot be predicted which additive is going to improve the PCR performance since the effect is very application dependent. In some cases, usage of one additive in two different PCR applications, can have opposing effects.

[0009] Betaine has among others, important applications in PCR and DNA sequencing. It has been described as a PCR enhancer, included as part of a standard optimization procedure. Betaine is known to disrupt secondary structures and to lower the melting temperature of the template improving template denaturation which is not only beneficial for PCR but also for Sanger sequencing; see Henke et al. (1997), Betaine improves the PCR amplification of GC-rich DNA sequences. Nucleic Acids Res. 25(19):3957-8; Rees et al. (1993), Betaine can eliminate the base pair composition dependence of DNA melting, Biochemistry 32(1):137-44; Shammas et al. (2001), Improvement of Quantitative PCR Reproducibility by Betaine as Determined by Fluorescence- Based Method. BioTechniques 30:950-954.

[0010] Betaine can be titrated either separately or in combination with DMSO; cf. Jensen et al. (2010), DMSO and betaine greatly improve amplification of GC-rich constructs in de novo synthesis, PLoS One 5(6):e11024; Kang et al. (2005), The enhancement of PCR amplification of a random sequence DNA library by DMSO and betaine: application to in vitro combinatorial selection of aptamers, J. Biochem. Biophys. Methods 64(2):147-51; Frackman et al. (1998), Betaine and DMSO: Enhancing Agents for PCR, Promega Notes 65:27. Betaine can improve target accessibility, enhance efficiency and specificity of DNA amplification and sequencing reactions.

[0011] The optimal concentration of betaine is described in the art as varying depending on the specific PCR or sequencing protocol, template and target sequence and must be experimentally determined. In most cases betaine is used with a finalconcentration of 0.5 - 2 M. However, it has been reported that even 2 M betaine is not sufficient for efficient amplification and / or sequencing of an AAV genome, cf. Liu et al. (2021), Disruptors, a new class of oligonucleotide reagents, significantly improved PCR performance on templates containing stable intramolecular secondary structures, Anal. Bi- ochem, 624:114169.

[0012] Further, it is important to note that the interference of betaine with the melting temperature does not only affect the template but also the binding dynamics of, e.g., primers and probes. The concentration of betaine that is needed to resolve the nucleic acid secondary structures allowing efficient nucleic acid amplification is too high so that it already negatively affects PCR performance.

[0013] Alternatives to betaine used to relieve the inhibitory effects of the ITR structures is to remove such ITR structures endonucleatically by, for example, restriction enzymes cutting within the ITR structures or usage of blocker oligos preventing the ITRs to form secondary structures; cf. Liu et al. (2021, loc. cit.), Prantner and Maar (2023), Genome concentration, characterization, and integrity analysis of recombinant adeno-associ- ated viral vectors using droplet digital PCR, PLoS One. 18(1):0280242. Use of restriction enzymes leads to significantly optimized quantifications without the need for adaptation of assays and PCR conditions. However, analysis of genome integrity and quality of capsid packaging is no longer possible which reduces the amount of information that can be achieved and that is an essential readout for evaluation of vector quality. Blocker oligos are very difficult to design since they must prevent ITRs to form without reducing PCR performance or cross-reacting with other target regions on the genome of interest.

[0014] Against this background, it is an object of the invention to improve existing methods for nucleic acid processing, such as PCR, sequencing and reverse transcription (RT), in such a way that even "tricky" nucleic acid molecules can be processed effectively and reliably. This applies in particular to those nucleic acid molecules that tend to develop secondary structures to a greater extent, such as, e.g., the genomes of AAVs. The aim is to avoid or at least reduce the disadvantages associated with the methods known in art.

[0015] The present invention satisfies these and other needs.SUMMARY OF THE INVENTION

[0016] This object underlying the invention is achieved by the use of a combination of betaine and magnesium chloride (MgCh) to improve nucleic acid processing.

[0017] The object is also met by a composition for nucleic acid processing comprising a combination of betaine and magnesium chloride (MgCh).

[0018] The object is also met by a method for nucleic acid processing, comprising the following steps:(1) Providing a composition comprising betaine and magnesium chloride (MgCh);(2) Adding nucleic acid to said composition, and(3) Processing said nucleic acid.

[0019] The properties, features, advantages and embodiments of the invention set forth in the following apply equally to the use, the composition and the method according to the invention.

[0020] The inventors have found that it is the combination of both compounds, betaine and MgCh, that leads to an improvement in nucleic acid processing. Thus, while betaine alone has an effect on the resolution of secondary structures, it inhibits, on the other hand, with increasing concentration, the binding to the nucleic acid, for example, of primers, probes or other factors. The inventors realized for the very first time that the addition of MgCh counteracts these negative effects of betaine. The inventors have also found that the combined presence of betaine and MgCh is able to mitigate and, if applicable, correct otherwise suboptimal amplification caused, for example, due to inhibitory components such as ethanol.

[0021] According to the invention, a "combination" is therefore understood to mean that both betaine and MgCh are brought into contact with the nucleic acid to be processed. This joint contacting can take place simultaneously, for example by providing both compounds in a common nucleic acid buffer in which the nucleic acid is incubated and / or processed.

[0022] According to the invention, "improvement of nucleic acid processing" means any optimization of the processing compared to reaction approaches without the added combination of betaine and MgCh, such as an increase in the efficiency, precision and accuracy of nucleic acid processing and quantification. This improvement can be illustrated in a digital polymerase chain reaction (dPCR), for example, by a reduction in the so- called "rain", i.e. , the number of positive partitions with lower fluorescence compared to the main population of positive partitions, where, in the art, it is unclear whether amplification with identical efficiencies has taken place in these partitions or not.

[0023] According to the invention, "nucleic acid processing" means the treatment of nucleic acids by means of common molecular biological and / or biotechnological methods, such as polymerase chain reaction (PCR), reverse transcription (RT) or sequencing, etc.

[0024] According to the invention, "nucleic acid" comprises deoxyribonucleic acid (DNA) and, in an embodiment, ribonucleic acid (RNA), each of human, animal, plant, bacterial, viral etc. origin.

[0025] The "use" according to the invention comprises the combined application of betaine and MgCh, for example, as two additives to or components of a solution commonly applied for processing nucleic acid, such as an aqueous solution, distilled water, saline, buffers (e.g., TE, TRIS, EDTA, TAE, HEPES, etc.).

[0026] The "composition" according to the invention can be present in solid, semi-solid, liquid or fluid form. In solid or semi-solid form, the betaine and the MgCh can each be present in powder, crystalline or lyophilized form. In liquid or fluid form, thebetaine and MgCh are present dissolved in a carrier, diluent or solvent capable of maintaining nucleic acid, containing said betaine and MgCh. Such carrier, diluent or solvent may include any of aqueous solution, distilled water, saline, buffers (e.g., TE, TRIS, EDTA, TAE, HEPES, etc.).

[0027] In embodiments of the invention, the composition comprises, consists or consists essentially of betaine and MgCh. I .e. , in the two latter embodiment, the composition according to the invention contains no or essentially no further compounds and / or salts, except betaine and MgCh. In other words, a liquid or fluid composition according to the invention, besides the carrier, diluent or solvent, such as aqueous solution, distilled water, saline, buffers, consists or essentially consists only of betaine and MgCh. "Essentially consists" in this context means that further components can be present, but only to the extent that these components do not materially affect the essential characteristics of the composition.

[0028] Betaine is any neutral chemical compound with a positively charged cationic functional group that bears no hydrogen atom, such as a quaternary ammonium or phosphonium cation and with a negatively charged functional group, such as a carboxylate group that may not be adjacent to the cationic site. All kinds of betaine turned out being suitable to carry out the invention. Examples of betaines particularly suitable according to the invention include, but are not limited to, the following compounds:, wherein Ri, R2, and R3 are H, CH3, C2H5 or any other alkyl, and R' is an amino acid residue; N,N,N-trimethylglycine (glycine betaine), N,N,N-trimethyl-betaine monohydrate.

[0029] The procedure described in this invention allows to reliably quantify nucleic acid targets that would be tricky to quantify because of the formation of secondary structures, such as AAV genome targets, in an accurate and precise manner using standard protocols simply by adding betaine and MgCh to the reaction.

[0030] Applications can be, for example, the amplification of long or coiled nucleic acid targets with higher processivity, discrimination of low and high GC content nucleic acid sequences or species in next generation sequencing (NGS) applications as well as the generation of cDNA in reverse transcription (RT) reactions.

[0031] The inventors' findings were surprising and not to be expected. For example, the prior art describes that betaine can be a PCR enhancer. However, it was not known that the addition of MgCh can reduce and possibly even prevent the negative effects of betaine, e.g., the reduced binding ability of PCR primers, probes or other factors to the nucleic acid to be processed.

[0032] The object underlying the invention is herewith fully achieved.

[0033] In a preferred embodiment of the invention said betaine is provided in a concentration in the range of 0.125 M to 5 M and said MgCh is provided in a concentration in the range of 0.65 mM to 23.25 mM. For the composition according to the invention this means that the composition contains betaine and MgCh in the indicated concentration ranges.

[0034] This measure has the advantage of providing such concentrations of betaine and MgCh that, according to the findings of the inventors, result in a particularly good improvement of nucleic acid processing.

[0035] According to the invention, a concentration range of 0.125 M to 5 M betaine includes the following concentrations (in M): 0.125, 0.25, 0.375, 0.5, 0.625, 0.75, 0.875, 1 , 1.125, 1.25, 1.375, 1.5, 1.625, 1.75, 1.875, 2, 2.125, 2.25, 2.375, 2.5, 2.625, 2.75, 2.875, 3, 3.125, 3.25, 3.625, 3.75, 3.875, 4, 4.125, 4.25, 4.625, 4.75, 4.875, 5, and respective intermediate concentrations.

[0036] According to the invention, a concentration range from 0.65 mM to 23.25 mM MgCh includes the following concentrations (in mM): 0.65, 0.652, 1.0, 1.375, 1.7,2.375, 2.7, 3.375, 3.7, 4.375, 4.7, 5.375, 5.7, 6.375, 6.7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23.25, and concentrations in between.

[0037] In the use according to the invention, the betaine and the MgCh can be provided in the medium or composition in which the nucleic acid processing takes place. The medium or composition can be, for example, a reaction medium, e.g., a nucleic acid buffer such as a PCR or sequencing or RT buffer or water or an aqueous solution, to which the betaine and the MgCh are added as additives or where the betaine and the MgCh are components.

[0038] The concentrations given afore are final concentrations in the medium or the composition according to the invention and take into account any amounts of betaine and MgCh already present in a composition or buffer, such as in a PCR master mix.

[0039] In yet another embodiment of the invention said nucleic acid processing is nucleic acid amplification.

[0040] This measure has the advantage of improving such a nucleic acid processing, which is particularly relevant in molecular biology but also in diagnostic practice. Nucleic acid amplification includes any method with which nucleic acid molecules, in particular DNA and / or RNA, can be amplified, such as any kind of polymerase chain reactions (PCR).

[0041] Accordingly, in another embodiment nucleic acid amplification is polymerase chain reaction (PCR), preferably digital PCR (dPCR).

[0042] With this measure, the invention is advantageously applied to particularly important nucleic acid amplification methods. Especially in dPCR, the amplification of nucleic acids that tend to form secondary structures, such as those with inverted terminal repeats (ITRs) and / or a high GC content, is a particular challenge. There are often partitions with lower fluorescence than the average positive partitions, so that it is unclear whether amplification has efficiently and / or specifically taken place in these partitions oronly inefficiently and / or unspecifically (so-called "rain"). This interfering effect can be particularly well eliminated or mitigated due to the invention.

[0043] In yet another embodiment the nucleic acid is nucleic acid sequencing.

[0044] In still another embodiment the nucleic acid processing is reverse transcription (RT).

[0045] The two exemplary embodiments mentioned above adapt the invention in an advantageous manner to nucleic acid processing methods which are particularly relevant in practice, and which have hitherto not provided satisfactory results in the case of nucleic acids with a tendency to form secondary structures. The invention provides a remedy here.

[0046] In another embodiment of the invention said nucleic acid, the processing of which can be improved by the invention, is GC-rich nucleic acid, preferably nucleic acid comprising a GC content of approx. >50%, preferably approx. >60%, preferably approx. >70%, further preferably approx. >80%.

[0047] Such GC-rich nucleic acids tend to develop secondary structures that make effective and reliable processing and amplification difficult. The invention provides an effective remedy here. The inventors have found that the betaine reduces the formation of the secondary structures by lowering the melting temperature Tm, whereas the MgCh further enables the primers, probes and any other necessary factors still to anneal or bind well to the nucleic acid.

[0048] In yet another embodiment of the invention said nucleic acid comprises inverted repeats (I s), preferably inverted terminal repeats (ITRs).

[0049] In still another embodiment of the invention said nucleic acid is adeno- associated virus (AAV) nucleic acid.

[0050] Nucleic acids comprising ITRs, or genomes, plasmids or vectors of adeno-associated viruses (AAVs), or nucleic acids derived from or originating from AAVs, express secondary structures to a particularly high degree and are therefore difficult to process with standard nucleic acid processing methods, such as PCR or sequencing. Here, too, the invention provides an effective remedy.

[0051] Another subject-matter of the invention is a kit for nucleic acid processing comprising betaine and magnesium chloride (MgCh), and a manual comprising instructions for nucleic acid processing.

[0052] The properties, features, advantages and embodiments of the use, the composition and the method according to the invention apply equally to the kit according to the invention.

[0053] In the kit the betaine and MgCh may be provided in separate containers or a common container, each in solution or in lyophilized and / or crystalline formulation, and / or as part of a preformulated master mix. The kit may comprise another container containing a diluent or reconstituting solution for the lyophilized and / or crystalline formulation. The manual comprises instructions for carrying out the nucleic acid processing methods or, alternatively, for pre-treating the nucleic acid before subjection it to a processing method.

[0054] The kit may further comprise nucleic acid, one or more of a buffer, a diluent, a filter, a needle, or a syringe. The container(s) is / are preferably a bottle, a vial, a syringe or test tube; and it may be a multi-use container. The container(s) may be formed from a variety of materials such as glass or plastic. Preferably the kit and / or container con- tain / s instructions on or associated with the container(s) that indicate directions for reconstitution and / or use. For example, the label may indicate that the lyophilized and / or crystalline formulation is to be reconstituted to salt concentrations as described above.

[0055] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art towhich this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the detailed methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided can be different from the actual publication dates, which can need to be independently confirmed.EMBODIMENTS

[0056] Before the present invention is further described, it is to be understood that this disclosure is not strictly limited to particular embodiments described herein, as such can of course vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the claims.

[0057] The invention is now further explained by means of embodiments resulting in additional features, characteristics and advantages of the invention. The features mentioned in the specific embodiments are features of the invention and may be seen as general features which are not applicable in the specific embodiment but also in an isolated manner in the context of any embodiment of the invention.

[0058] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0059] The invention is now further described and explained in further detail by referring to the following non-limiting examples and figures:Fig. 1 : Betaine affects amplification and quantification in a concentration and tar- get-dependent manner. AAV2 (supplier Vector Biolabs, cat. no. 7004) lysates were produced using the CGT Viral Vector Lysis kit and stored at -20 °C. The lysate was thawed right before use, added to the QIAcuity Probe MM containing different concentrations of betaine. Hpall restriction enzyme was added to the PCR reaction, incubated for 10 minutes at RT before transfer of reaction to an 8.5k nanoplate. Quantification was performed using the CGT dPCR assays in a triplex reaction (A: CMV promoter- FAM, B: ITR-HEX, D: CMV enhancer-Cy5) following the recommended cycling and imaging conditions. A, B, C: 1 D scatterplots shown for all assays. D: Target quantification normalized to the quantification in a PCR reaction without betaine (100%). Mean of 3 replicates per condition shown.Fig. 2: Fig. 2 Betaine affects amplification and quantification in a concentration and target-dependent manner. AAV5 (supplier Vector Biolabs, cat. no. 7006) lysates were produced using the CGT Viral Vector Lysis kit and stored at -20 °C. The lysate was thawed right before use, added to the QIAcuity Probe MM containing different concentrations of betaine. Hpall restriction enzyme was added to the PCR reaction, incubated for 10 minutes at RT before transfer of reaction to an 8.5k nanoplate. Quantification was performed using the CGT dPCR assays in a triplex reaction (A: CMV promoter- FAM, B: ITR-HEX, C: CMV enhancer-Cy5) following the recommended cycling and imaging conditions. A, B, C: 1 D scatterplots shown for all assays. D: Target quantification normalized to the quantification in a PCR reaction without betaine (100%). Mean of 3 replicates per condition shown.Fig. 3: Betaine and MgCl2 together can improve amplification of tricky targets.AAV2 (unpurified, inhouse produced) samples were processed using the CGT Viral Vector Lysis kit. The lysates were added to the QIAcuity Probe MM containing different concentrations of betaine and MgCL Hpall restriction enzyme was added to the PCR reaction, incubated for 10 minutes at RT before transfer of reaction to an 8.5k nanoplate. Quantification was performed using the CGT dPCR assays in a multiplex reaction (A1 : SV40 promoter-FAM, A2: SV40pA-Cy5) following the recommended cycling and imaging conditions. A1 , A2: 1 D scatterplots shown for all assays. B: Target quantification normalized to the quantification in a PCR reaction without additives (100%). Mean of 3 replicates per condition shown.Fig. 4: Optimal betaine and MgCl2 concentrations need to be titrated in a targetdependent manner. AAV2 (unpurified, inhouse produced) samples were processed using the CGT Viral Vector Lysis kit. The lysates were added to the QIAcuity Probe MM containing different concentrations of betaine and 3.375 mM MgCh Hpall restriction enzyme was added to the PCR reaction, incubated for 10 minutes at RT before transfer of reaction to an 8.5k nanoplate. Quantification was performed using the CGT dPCR assays in a multiplex reaction (SV40 promoter-FAM, SV40pA-Cy5) following the recommended cycling and imaging conditions. Mean target quantification of 3 replicates per condition shown.Fig. 5: Defined concentrations of betaine and MgCl2 make use of restriction enzymes for AAV genome titration redundant. AAV2 (unpurified, inhouse produced) samples were processed using the CGT Viral Vector Lysis kit. The lysates were added to the QIAcuity Probe MM containing different concentrations of 1.375 M betaine and different MgCl2 concentrations ranging from 0.624 mM to 6.25 mM. Hpall restriction enzyme was only added to the control reaction that served as a reference. All other samples were not treated with a restriction enzyme. All samples (with and without restriction enzyme) were incubated for 10 minutes at RT before transfer of reaction to an 8.5k nanoplate. Quantification was performed using the CGT dPCR assays in amultiplex reaction (A1 : SV40 promoter-FAM, A2: ITR-HEX, A3: SV40pA- Cy5) following the recommended cycling and imaging conditions. A1 , A2, A3. 1 D- scatterplots shown. B. Mean target quantification of 3 replicates per condition for SV40 promoter and SV40pA shown. C. 1 D-scatterplots comparing a standard condition with and without restriction digest as well as the PCR condition with 1.375 M betaine and 5 mM MgCl2 without a restriction digest are shown.Fig. 6: Defined concentrations of betaine and MgC make use of restriction enzymes for AAV genome titration redundant. AAV2 (unpurified, inhouse produced) samples were processed using the CGT Viral Vector Lysis kit. The lysates were added to the QIAcuity Probe MM containing different concentrations of 1.375 M betaine and different MgCl2 concentrations ranging from 5 mM to 6.25 mM. Hpall restriction enzyme was only added to the control reaction that served as a reference. All other samples were not treated with a restriction enzyme. All samples (with and without restriction enzyme) were incubated for 10 minutes at RT before transfer of reaction to an 8.5k nanoplate. Quantification was performed using the CGT dPCR assays in a multiplex reaction (SV40 promoter-FAM, SV40pA-Cy5) following the recommended cycling and imaging conditions. A1 , A2, A3. 2 D-scatterplots shown. B. Mean target quantification of 3 replicates per condition for SV40 promoter and SV40pA shown.Fig. 7: 1 D plot showing the relative fluorescence Units of the SV40 pA assay without any additions (first column) and increasing MgCh concentrations in the presence of 1.5 M betaine.Fig. 8: 1 D Plot showing a high GC target with and without additional betaine andMgCh. The presence of Q-Solution alone improved the quantification and formation of a positive cluster (second column). Further addition of MgCh lead to positive cluster formation with little rain (rightmost 4 columns).ExamplesConcept

[0060] Genome titer determination of AAV samples might be challenging under PGR conditions due to the complex ITR structures with high GC-content and stable secondary structures reducing accessibility to the targets of interest.

[0061] Aim: Optimization of PGR reaction conditions in order to get:1. Reduction of "rain" (positive partitions with lower RFUs compared to the main population of positive partitions).2. Target quantification comparable to genomes treated with restriction enzymes removing challenging ITR structures.3. Consistent PCR performance / efficiency in all partitions of one well.

[0062] To achieve accurate genome vector titers, betaine and MgCh were titrated. Optimal concentrations made use of restrictions enzymes, targeting the ITR structures, redundant.Addition of betaine does slightly reduce number of positive partitions with lower fluorescence and does not significantly improve quantification

[0063] In order to improve quantification of AAV targets encoded by AAV2 and AAV5 particles and to reduce rain, different concentrations ranging from 0.5 M to 2 M betaine were added to the QI Acuity Probe MM. AAV2 and AAV5 samples were processed using the CGT Viral Vector Lysis kit following the manufacturers recommendations (QIAGEN, cat. no. 250272). The lysates were stored at -20°C and thawed right before use at RT. Afterwards, the lysates (1.2 pl per 12 pl reaction) were added to the QIAcuity Probe MM containing 0.025 II Hpall restriction enzyme (Thermo Fisher ANZA, IVGN0936) mixedthoroughly and incubated for 10 minutes at RT. ITR, CMV promoter and CMV enhancer regions were targeted using the CGT dPCR assays in a triplex reaction using the FAM, HEX and Cy5 channel.

[0064] After incubation, the PCR mixes were transferred to an 8.5k nanoplate and analyzed on a QIAcuity dPCR system following the recommended cycling and imaging conditions.

[0065] Under standard conditions (QIAcuity Probe MM without betaine), amplification of the CMV promoter and CMV enhancer was not consistently efficient throughout all partitions of a well which is reflected by positive partitions with lower fluorescence especially in the middle of a well. Addition of 0.5 M betaine (N,N,N-trimethyl-betaine monohydrate) already improved PCR performance (Fig. 1, Fig. 2).

[0066] Reduction of "rain" compared to the standard reaction without betaine could be observed in AAV2 and AAV5 samples in reactions supplemented with 0.5 M betaine for all three targets ITR, CMV promoter and CMV enhancer. However, increased concentrations of betaine led to a slight increase of "rain". Addition of 2 M betaine negatively affected PCR performance. Number of partitions of lower fluorescence significantly increased as well as the quantification significantly decreased. The 3 targets of interest were all differently impacted by the addition of 2 M betaine with the CMV enhancer showing the highest drop in quantified titer (Fig. 1, Fig. 2).

[0067] As described in multiple publications, betaine serves as a PCR enhancer in a concentration and target-dependent manner.

[0068] The analysis of different AAV2 samples (unpurified, produced inhouse) encoding the targets SV40 promoter and SV40pA showed that addition of 2 M betaine was beneficial for the amplification of the SV40 promoter (reduced rain in 1 D-scatterplotsand increased quantification compared to the standard QIAcuity Probe MM without betaine) but not for the amplification of SV40pA (Fig. 3).

[0069] Betaine reduces the melting temperature of the template facilitating to resolve secondary structures and improving accessibility of target regions. However, betaine also affects the melting characteristics of primers and probes to different extents since among others, the assay sequences play an important role. Another important component of a PCR mix is MgCh. MgCh acts as a cofactor enhancing enzymatic activity of DNA polymerases.

[0070] To investigate the interplay of betaine and MgCh and to see if MgCh can counteract the reduced PCR performance of the SV40pA amplification, 3.375 mM and 6.7 mM MgCh was added to PCR reactions containing 2 M betaine. MgCh was able to counteract the negative effect of 2 M betaine on the amplification of SV40pA without negatively affecting the quantification of the SV40 promoter target. MgCh without betaine did not improve SV40pA amplification. Both additives betaine and MgCh are needed to enable accurate and precise quantification of various targets (Fig. 3).

[0071] To further investigate the interplay of MgCh with different betaine concentrations, AAV2 samples were prepared as described above and analyzed via dPCR on 8.5k nanoplates using the QIAcuity Probe mix with 3.375 mM MgCh. The betaine concentration was titrated from 0.125 M up to 2 M. The quantification of the SV40 promoter and SV40pA targets were monitored using CGT dPCR assays in a multiplex reaction. Quantification of SV40 and SV40pA was compared to the titer obtain running a PCR without additives (Fig. 4). The SV40 promoter quantification was already increased under addition of 0.5M betaine and 3.375 mM MgCh. Under those PCR conditions, the quantification of SV40pA remained unchanged. 1 M betaine and 3.375 mM MgCh further increased the quantification of SV40 promoter but not SV40pA. Addition of 2 M betaine with 3.375 mM MgCh increased both the quantification of SV40 promoter and SV40pA to the same level.

[0072] This indicates that higher levels of betaine and MgCh are needed to allow for accurate quantification of SV40pA without negatively affecting the quantification of the SV40 promoter target (Fig. 4).Optimal betaine and MgCI2concentrations can replace the function of a restriction enzyme in the titration of AAV genomes containing ITRs

[0073] For the application of AAV genome titer determination the secondary structures of the ITRs interfere with target accessibility leading to reduced quantification and inefficient PCR. To overcome that, in most cases restriction enzymes such as Hpall or Mspl removing the ITR secondary structures are used.

[0074] In order to analyze if betaine and MgCh are able to improve dPCR performance to the same extent as a restriction digest, AAV2 samples were processed as described above. The lysates were analyzed in a QIAcuity Probe MM with and without Hpall. The PCR mix was supplemented with 1.375 M betaine. The MgCh concentration was titrated from 0.652 mM up to 6.25 mM. The targets SV40 promoter and SV40pA were quantified using CGT dPCR assays in multiplex reactions.

[0075] The quantification of both targets was significantly reduced in the reaction without additives and without restriction enzyme. The underquantification could be resolved by adding betaine and MgCh (Fig. 5, Fig. 6). Addition of 1.375 M of betaine and 3.75 mM MgCh or higher led to comparable titers without the need to physically remove the ITR secondary structures with a restriction enzyme. Within the range of 3.75 mM and 6.25 mM MgCh the quantification of the SV40 promoter and SV40pA targets remained the same. The quantification results of the SV40pA target indicates that higher MgCh concentrations are needed compared to the SV40 promoter (Fig. 5).

[0076] The quantification of the ITR target was improved as well. The "rain" in the 1 D-scatterplots could be reduced by adding betaine and MgCh (Fig. 5). Reduction of rain and increased separation of positive and negative partitions could also be observed in the 2D-scatterplot of the SV40 promoter target and the SV40pA target. The separation ofthe population was even higher with 1.375 M betaine and 5 mM supplemented MgCh compared to the standard reaction containing no additives but incubated with the restriction enzyme Hpall (Fig. 6).PCR assays amplifying DNA with a low percentage of G and C nucleotides

[0077] The low GC % assay targeting SV40pa was run on a plasmid containing the SV40pa DNA sequence and the high GC% assay targeting the ITR sequence of the AAV2 genome was also run on a plasmid containing the ITR sequence. For both plasmids -1000 copies I pl

[0078] The low GC % assay was run with the QIAcuity probe Mastermix and an appropriate Taqman assay. MgCh was titrated in increasing concentrations (0, 2.5, 5 and 7.5 mM) in the presence of 1.5 M betaine. A reaction without additional betaine and MgCh served as a reference point.

[0079] The high GC % assay was run with the QIAcuity probe Mastermix and an appropriate Taqman assay. MgCh was titrated in increasing concentrations (0, 2.5, 5 and 7.5 mM) in the presence of 2 M Betaine. A reaction without additional betaine and MgCh served as a reference point.

[0080] The inventors tested PCR assays amplifying DNA with a low percentage of G and C nucleotides. When tested without Betaine and additional MgCh, the positive cluster showed -40 RFU. When adding 1.5 M of betaine and no additional MgCh, the positive cluster dropped 10 RFU to -30 (Fig. 7)

[0081] After addition of at least 2.5 mM MgCh into the 1.5 M Betaine reaction, the positive cluster could be restored to its original value of -40RFU. Moreover, adding more MgCh further boosted the signal (Figurel, right set of columns), while the absolute quantification of the target sequence slightly increased from -700 to around 800 cp / pl (Table 1 left column).

[0082] The results for the GC rich ITR assay are shown in Fig. 8. The addition von 2 M Betaine alone leads to a more efficient PGR reaction (less rain and a slight formation of a positive cluster) compared to the reaction without any additions. Increasing the MgCh concentration resulted in a more consistent PGR reaction and the formation of a positive cluster (Fig. 8, second column). Albeit the End RFU of the reaction was lower compared to the reference concentration, the quantification shows an increase from 200 to 400 copies I pl reaction in the presence of 2 M Betaine alone (Table 1 right column) compared to the reaction without additional Betaine. The results increased further in the presence of additional MgCh. Fig. 8 shows the formation of positive clusters with only little rain and an increase of copies I pl from 400 with only Betaine to -1000 which was expected based on the input amount (Table 1 right column).Table 1

Claims

Claims1. A use of a combination of betaine and magnesium chloride (MgCh) to improve nucleic acid processing.

2. The use of claim 1, wherein said betaine is provided in a concentration in the range of 0.125 M to 5 M and said MgCh is provided in a concentration in the range of 0.65 mM to 23.25 mM.

3. The use of claim 1 or 2, wherein said nucleic acid processing is nucleic acid amplification, preferably comprising polymerase chain reaction (PCR), further preferably digital PCR (dPCR).

4. The use of claim 1 or 2, wherein said nucleic acid processing comprises nucleic acid sequencing.

5. The use of claim 1 or 2, wherein said nucleic acid processing comprises reverse transcription (RT).

6. The use of any of the proceeding claims, wherein said nucleic acid is GC-rich nucleic acid, preferably nucleic acid comprising a GC content of approx. >50%, preferably >60%, preferably >70%, further preferably >80%.

7. The use of any of the proceeding claims, wherein said nucleic acid comprises inverted repeats (I Rs), preferably inverted terminal repeats (ITRs).

8. The use of any of the proceeding claims, wherein said nucleic acid is adeno-associ- ated virus (AAV) nucleic acid.

9. A composition for nucleic acid processing comprising a combination of betaine and magnesium chloride (MgCh).

10. The composition of claim 9, comprising betaine in the range of 0.125 M to 5 M and MgCh in the range of 0.65 mM to 23.25 mM.

11. The composition of claim 9 or 10, wherein said nucleic acid processing is nucleic acid amplification, preferably comprising a polymerase chain reaction (PCR), further preferably digital PCR (dPCR).

12. The composition of claim 9 or 10, wherein said nucleic acid processing comprises nucleic acid sequencing and / or reverse transcription (RT).

13. A kit for nucleic acid processing comprising betaine and magnesium chloride (MgCh), and a manual comprising instructions for nucleic acid processing.

14. A method for nucleic acid processing, comprising the following steps:(1) Providing a composition comprising betaine and magnesium chloride (MgCh);(2) Adding nucleic acid to said composition, and(3) Processing said nucleic acid.

15. The method of claim 14, wherein said composition comprises betaine in the range of 0.125 M to 5 M and MgCh in the range of 0.65 mM to 23.25 mM.

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