Method of detecting methylated nucleic acids

Abstract

The present invention relates to methods of detecting methylated nucleotides comprised by a nucleic acid molecule and to a kit adapted for carrying out said methods.

Description

Method of detecting methylated nucleic acids

[0001] The present invention relates to methods of detecting methylated nucleotides comprised by a nucleic acid molecule and to a kit adapted for carrying out said methods.FIELD OF THE INVENTION

[0002] The present invention relates to the field of molecular biology, more particularly to the detection of methylated nucleic acids.BACKGROUND OF THE INVENTION

[0003] Regulation of gene expression is a tightly controlled process, mandatory for cell function. One possibility to regulate gene expression is termed methylation and commonly refers to the attachment of a methyl group (CH3) to a cytosine nucleotide. A methylatable cytosine usually occurs in combination with other nucleotides, e.g. with a neighboring guanine. A single site is termed a CpG site, meaning a dinucleotide (cytosine- phosphate-guanine) of which the cytosine is then modified. In most cases, several CpG sites are located in close proximity forming a CpG "island". The degree of methylation of these islands, i.e. the number of methylated CpG sites, can be used by a cell to regulate gene expression. Due to the nature of several CpG sites being in close proximity, determining the methylation status or degree of methylation of an island using PCR can be difficult as primers / probes can be difficult to design.

[0004] Several ways to detect the methylation status of a DNA have been developed. The prerequisite for nearly all of them is bisulfite conversion of DNA. In this step, all unmethylated cytosine nucleotides are chemically being changed to uracil nucleotides by way of deamination leaving methylated cytosine nucleotides unchanged. In the resulting DNA sequence, the presence of a cytosine hence reflects the presence of a methylation site.

[0005] Since most potential methylation sites in the genome are well characterized, assays specific to certain sites can be designed.

[0006] One of the first approaches to investigate methylation using PCR was methylation specific PCR (MSP). In MSP the primers used for normal endpoint PCR were designed to match the bisulfide modified DNA sequence when at least one CpG site was initially methylated. The primer covering the CpG site would then only bind when a methylation was present and lead to a detectable PCR product; see Huang et al. (2013), Methylation-Specific PCR, Methods in Molecular Biology, Vol. 1049, 75-82.

[0007] Later methylation specific TaqMan probes, for example used in Qiagen's MethyLight assay system, have been used. Here, two sequences specific DNA fragments (differently labelled TaqMan probes) are hybridized to the methylation site, one specific forthe methylated case (binding to a cytosine) and one for the unmethylated version binding specifically to a uracil in this position (instead of an cytosine).

[0008] In reality, both approaches are difficult to design due to the organization of methylation sites. In most cases, these sites are clustered together and appear in short distances, forming so called "methylation islands" making it very difficult to design primers and or probes specific for a single site. Having multiple sites within one assay makes it very difficult to determine how many sites were methylated in the original DNA using quantitative PCR. In addition, both approaches rely on known standards that are being rung in parallel, allowing to infer the methylation status based on comparison to such standards.

[0009] Next came pyrosequencing, a method where a single primer was used as a starting point and the DNA sequence was well characterized. After the initial primer bound to the DNA, individual nucleotides (C, T, A and G) were added to the PCR reaction, incorporated and residual nucleotides washed away in a pre-programmed sequence. When a methylated cytosine is present in the DNA sequence, a positive signal can be detected when a matching Guanin was added to the reaction; see Nolan and Bustin (Eds.) (1994), Pyrosequencing Technology in "PCR Technology: Current Innovations", 168-175. The problem with the detection is when there is a stretch of the same nucleotides, incomplete washing steps and a cumbersome process.

[0010] With the dawn of next generation sequencing, researchers quickly adapted the technology to look for methylation events. Here, either the whole genome or a (or several) complete CpG island is being amplified and individual copies sequenced in mass and in parallel; Zhang and Jeltsch et al. (2010), The Application of Next Generation Sequencing in DNA Methylation Analysis, Genes 1(1), 85-101. As with normal sequencing, NGS is still, compared to digital PCR, a cumbersome and costly approach which requires a lot of technical expertise and has an inherent long turn-around time.

[0011] Digital PCR (dPCR), most notably the Bio-Rads QX200 system, has been used to determine methylation status of single CpG site using the MethyLightapproach; see Tai et al. (2018), DNA Methylation Analysis Using Droplet Digital PCR in "Digital PCR: Methods and Protocols", 363-383, and US 2015 / 0099266. However, assay design and data interpretation can be difficult depending on the specific DNA sequence.

[0012] A dPCR-based method to determine methylated DNA is also disclosed in WO 2023 / 129965.

[0013] Against this background it is an object underlying the invention to provide a method of detecting methylated nucleotides by means of which the disadvantages of the methods of the art are improved or even avoided.

[0014] The present invention satisfies these and other needs.SUMMARY OF THE INVENTION

[0015] This problem underlying the invention is solved by a method of detecting methylated nucleotides comprised by a nucleic acid molecule comprising the following steps: a) Providing a nucleic acid molecule suspected of comprising methylated nucleotides; b) Providing a methylation detection probe configured to specifically bind to methylatable nucleotides of said nucleic acid molecule, said specific binding to said methylatable nucleotides in non-methylated state occurs with a reference binding efficiency value VR; c) Incubating said nucleic acid molecule and said methylation detection probe under conditions allowing the specific binding of said methylation detection probe to said nucleic acid molecule, and d) Determining a value VA of an actual binding efficiency of the methylation detection probe to the nucleic acid molecule, e) Comparing A and VR, andf) Detecting methylated nucleotides if VA < VR.

[0016] The inventors have found that the detection of methylated nucleotides in a target nucleic acid molecule is possible by using a specific methylation detection probe which binds to the nucleic acid molecule with varying degrees of affinity depending on the degree of methylation of the nucleotides of the target nucleic acid molecule. In the case of absent or low methylation, i.e. a low degree of methylation, the methylation detection probe binds to the nucleic acid molecule with high affinity, i.e. high binding efficiency. In the case of strong methylation, i.e. a high degree of methylation, the methylation detection probe binds to the nucleic acid molecule with low affinity, i.e. low binding efficiency. The comparison of the measured, actual binding efficiency VA with a reference value R, which represents the - high - binding efficiency of the methylation detection probe in the absence of methylation, makes it possible to determine whether the nucleic acid molecule is methylated or not. If the measured, actual binding efficiency VA is lower than the reference value VR, methylation is present; if the measured, actual binding efficiency VA is at least equal to the reference value VR, no methylation is present.

[0017] It goes without saying, that the nucleic acid molecule suspected of comprising methylated nucleotides can be provided in step (a) in a sample, such as a liquid sample, i.e. in solution such as an appropriate buffer well known to the skilled person.

[0018] "Nucleic acid molecule", "nucleic acid" or "polynucleotide", as used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase, or by a synthetic reaction. Thus, for instance, nucleic acid molecules as defined herein include, without limitation, single- and double-stranded DNA, DNA including single- and double-stranded regions, single- and double-stranded RNA, and RNA including single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or include single- and double-stranded regions.

[0019] "Methylatable nucleotides" refer to specific nucleotides or nucleotide sequences within a nucleic acid molecule that are capable of undergoing methylation, a biochemical process involving the addition of a methyl group (-CH3) to a nucleotide. Methylation most commonly occurs at the 5' carbon of the cytosine ring in the context of a cyto- sine-guanine dinucleotide (CpG), but can also occur at other dinucleotide sequences such as CpA, CpT, and CpC. CpG (cytosine-phosphate-guanine) is a dinucleotide sequence where a cytosine (C) is followed by a guanine (G) with a phosphate (p) linkage between them. The cytosine residue in CpG can be methylated to form 5-methylcytosine, which is a common epigenetic modification affecting gene expression. CpA (cytosine-phosphate- adenine) is a dinucleotide sequence where a cytosine (C) is followed by an adenine (A) with a phosphate (p) linkage between them. This sequence can also be a target for methylation at the cytosine residue, although less frequently than CpG. CpT (cytosine-phos- phate-thymine) refers to a dinucleotide sequence where a cytosine (C) is followed by a thymine (T) with a phosphate (p) linkage between them. Methylation can occur at the cytosine residue within this sequence. CpC (cytosine-phosphate-cytosine) is a dinucleotide sequence where a cytosine (C) is followed by another cytosine (C) with a phosphate (p) linkage between them. Both cytosine residues can potentially be methylated. "Methylatable nucleotides" can be arranged in so-called islands. An "island" of methylatable nucleotides is a region of DNA that has a high frequency of methylatable dinucleotides relative to the rest of the genome. The most common island is the "CpG island", which is a region of DNA that has a high frequency of CpG dinucleotides relative to the rest of the genome. These islands are typically 300 to 3,000 base pairs in length and are often located near or within the promoter regions of genes. While the term "CpG island" is the most common, similar concepts can apply to regions rich in other methylatable dinucleotides such as CpA, CpT, and CpC, although these are less frequent.

[0020] "Specific binding" refers to the selective and precise interaction between a nucleotide probe, such as the 'methylation detection probe', and the (target) nucleic acid molecule, based on the sequence complementarity between the probe and the target. This interaction is primarily governed by the principle of base pairing, where adenine (A) pairs with thymine (T) or uracil (II), and cytosine (C) pairs with guanine (G). The concept of complementarity is central to specific binding. A nucleotide probe is designed to have asequence that is complementary to a specific region of the target nucleic acid molecule. The degree of complementarity directly affects the specificity of the binding.

[0021] The "methylation detection probe" provided in step (b) is configured to specifically bind to methylatable nucleotides of the target nucleic acid molecule with high binding efficiency or high affinity, respectively in case said methylatable nucleotides are non-methylated. "Reference binding efficiency value" or "VR" refers to a quantitative measure or numerical value of the binding efficiency of the methylation detection probe to the target nucleic acid molecule when the methylatable nucleotides are in their non-methyl- ated state. VR represents the maximum binding efficiency (e.g., 1.0), which decreases when the methylatable nucleotides are methylated.

[0022] The "conditions" in step (c) allowing the specific binding of said methylation detection probe to said nucleic acid molecule are well known to the skilled person. They refer to the set of physical, chemical, and environmental parameters that must be controlled and maintained to ensure that the methylation detection probe binds specifically and effectively to the methylatable nucleotides of the target nucleic acid molecule. These conditions include, e.g., temperature, pH, ionic strength, buffer composition, probe and nucleic acid concentrations, washing conditions etc. An example of standard hybridization conditions is: temperature = 55°C; pH = 7.5; ionic strength = 0.3 M NaCI, buffer = 20 mM Tris-HCI, 1 mM EDTA, probe concentration = 10 nM, incubation time = 1 hour, washing: 2x SSC (saline-sodium citrate buffer) at 60°C. Another example of high-stringency hybridization conditions include: temperature = 60°C; pH = 8.0; ionic strength = 0.1 M NaCI; buffer = 50 mM Tris-HCI, 2 mM EDTA, 50% formamide; probe concentration = 5 nM; incubation time = 2 hours; washing = 0.1x SSC at 65°C.

[0023] Value "VA" is a numerical value and refers to the actual binding efficiency of the methylation detection probe to the nucleic acid molecule under current experimental conditions. This value reflects the effectiveness with which the probe hybridizes to the methylatable nucleotides of the target nucleic acid molecule, taking into account the presence or absence of methylation at the methylatable nucleotides. When the methylation degree is low, VA is high (e.g., 0.8), indicating strong and specific binding of the probe to the non-methylated target sequence. When the methylation degree is high, VA is low (e.g.0.2), indicating reduced binding efficiency of the probe due to the presence of methyl groups. VA corresponds to the reference binding efficiency value VR when there is no methylation present (e.g., 1.0), representing the maximum binding efficiency of the probe. This measure allows for the assessment of methylation status by comparing actual binding efficiency to the reference efficiency.

[0024] In step (e) VA and R are compared and in step (f) a methylation of the methylatable nucleotides can be confirmed or detected if the methylation detection probe binds with lower efficiency that the reference value, i.e. with a VA that is lower than VR.

[0025] In an embodiment of the method according to the invention after step (a) and before step (b) the following step is carried out: a') subjecting the nucleic acid molecule to a bisulfite conversion reaction.

[0026] This measure has the advantage of using a method that converts the chemical modification of an attached methyl group into sequence information that can be easily detected using conventional hybridization techniques.

[0027] "Bisulfite conversion reaction" refers to a chemical treatment process used to detect methylated nucleotides within a nucleic acid molecule. During this reaction, all unmethylated cytosine nucleotides (C) are chemically converted to uracil nucleotides (II) through a process called deamination, while methylated cytosine nucleotides remain unchanged. Cytosines that are methylated (5-methylcytosine) are resistant to bisulfite-in- duced deamination and remain as cytosine in the sequence. Consequently, in the resulting nucleotide sequence, the presence of uracil (read as thymine (T) in sequencing) indicates an originally unmethylated cytosine, whereas the presence of cytosine indicates a methylated cytosine. This method allows for the specific detection of methylation at singlebase resolution, making it highly sensitive and accurate for mapping methylation patterns. Bisulfite conversion can be used in conjunction with various techniques such as polymerase chain reaction (PCR). In this embodiment, methylation detection probes are designedto specifically bind to the converted sequence. For example, probes targeting unmethylated regions must recognize the converted uracil (read as thymine).

[0028] In an embodiment of the method according to the invention in step (c) said nucleic acid molecule and said methylation detection probe are subjected to a polymerase chain reaction (PCR).

[0029] This measure has the advantage of using a method that is well established in laboratories and is particularly suitable for the detection and visualization of methylated nucleic acid molecules.

[0030] The technique of "polymerase chain reaction" or "PCR" as used herein generally refers to a procedure wherein minute amounts of a specific piece of nucleic acid, RNA and / or DNA, are amplified as described in the art, for example, in U.S. 4,683,195. Generally, sequence information from the ends of the region of interest or beyond needs to be available, such that oligonucleotide primers can be designed; these primers will be identical or similar in sequence to opposite strands of the template to be amplified. The 5' terminal nucleotides of the two primers may coincide with the ends of the amplified material. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, bacteriophage, or plasmid sequences, etc. See generally Mullis et al., Cold Spring Harbor Symp. Quant. Biol. 51 :263 (1987) and Erlich, ed., PCR Technology (Stockton Press, NY, 1989).

[0031] In an embodiment of the invention said PCR is digital PCR (dPCR).

[0032] This measure has the advantage that a variant of PCR is used that can be used to determine the degree of methylation of the target nucleic acid molecule particularly well and precisely.

[0033] Digital polymerase chain reaction (dPCR) is a biotechnological refinement of conventional polymerase chain reaction methods that can be used to directly quantify and clonally amplify nucleic acids strands including DNA or cDNA, but also RNA.dPCR offers certain advantages of conventional PCR. In dPCR, samples are partitioned in thousands of individual reactions prior to a PCR cycling reaction. This effectively decreases the concentration of target molecules within each individual partitioned PCR reaction, in contrast to traditional PCR where a bulk sample is being analyzed. End-point PCR then occurs in each of the partitions independently, producing a signal only in partitions where the target of interest is present. Ideally, only one nucleic acid molecule is being amplified in one partition. This leads to the effect that different PCR efficiencies result in different VA values and, therefore, different clusters. Due to the small volume the reaction is more tolerant towards contaminants or PCR inhibitors that might be introduced by the analyzed sample. dPCR detects PCR signals from individual partitions and calculates the absolute amount of target molecules present using Poisson statistics, eliminating the need for a standard curve. Due to these features, dPCR allows for absolute quantification of target molecules with a higher sensitivity and specificity compared to qPCR, down to a single target molecule. In addition, dPCR removes the challenges associated with bioinformatic analysis and interpretation of NGS data, is easily scalable to run in cost- efficient manner, doesn’t need overly expensive machines or reagents and has a fast turn-around time.

[0034] In an embodiment of the invention said methylation detection probe comprises a detectable marker configured to allow determination of VA.

[0035] This measure has the advantage that a methylation or the degree of methylation of the methylatable nucleotides can be determined via the visualization of the marker. The detectable marker can be any molecule or compound that can be detected by conventional methods.

[0036] Detectable markers that can be activated by PCR, including dPCR, are preferred. "PCR-activatable" markers are those that emit a signal when a PCR reaction takes place, i.e. the target nucleic acid molecule is amplified via PCR. An example of a marker that can be activated by PCR is the fluorophore / quencher pair, as used in TaqMan probes. Therefore, in an embodiment the methylation detection probe is configured as a TaqMan probe. Another example is the mediator sequences and universal reporters sytem, as used in the 'Mediator Probe PCR'; see Faltin et al. (2012), Mediator probe PCR: a novel approach for detection of real-time PCR based on label-free primary probes andstandardized secondary universal fluorogenic reporters, Clinical Chemistry. 58(11):1546- 56.

[0037] Furthermore, for this reason in an embodiment of the invention the detectable marker is a fluorescence marker, and VA and VR are represented by relative fluorescence units (RFU).

[0038] This measure provides the constructive prerequisites for reliable detection of methylation by means of visualization.

[0039] "Relative fluorescence units" (RFU) is a measure of the intensity of fluorescence emitted from a sample in a fluorescence-based assay. RFU values are used to quantify the amount of fluorescent signal, which is indicative of the presence and concentration of a specific target molecule or the activity of a particular process within the sample.

[0040] In one embodiment, the less methylated the methylatable nucleotides on the target nucleic acid molecule are, the stronger the fluorescence (or the higher the RFU) triggered or activated by the PCR is, and the more methylated the methylatable nucleotides on the target nucleic acid molecule are, the weaker the fluorescence (or the lower the RFU) is. The strength of the fluorescence or the RFUs are thus a direct measure of the binding efficiency of the methylation detection probe to the methylatable nucleotides of the target nucleic acid molecule and thus a direct measure of the degree of methylation of the methylatable nucleotides.

[0041] Examples for detectable, fluorescence markers include 6-carboxyfluo- rescein (FAM) and "hexachlorofluorescein" (HEX). In TaqMan probes said dyes are provided at the 5' end of the probe, and the quenchers are provided at the 3' end of the probe. Examples for suitable quenchers to be used with FAM include Black Hole Quencher-1 (BHQ-1), TAMRA (tetramethylrhodamine), Iowa Black FQ, Quenchers used with HEX include Black Hole Quencher-2 (BHQ-2), DABCYL (4-(4'-dimethylaminophe- nylazo)benzoic acid), and Iowa Black RQ.

[0042] In an embodiment of the invention VA comprises multiple distinct values (VAI , VA2, VAS, VAO) each representing a specific pattern of methylated nucleotides comprised by said nucleic acid molecule.

[0043] This embodiment takes into account the phenomenon that the methylatable nucleotides in the target nucleic acid molecule may be methylated to varying degrees, wherein 'ri is an integer between 1 and the maximum number of potential methylation sites in the target nucleic acid molecule. For example, if the target nucleic acid molecule has 4 methylatable nucleotides, i.e. 4 potential methylation sites, then a first distinct, strong fluorescence signal VAI can be measured in a completely unmethylated state (all 4 methylation sites are unmethylated). A second, slightly reduced fluorescence signal VA2 is obtained when 1 of the methylation sites is methylated and 3 of the methylation sites are unmethylated. A third, further reduced fluorescence signal VA3 is detected when 2 of the methylation sites are methylated and the remaining 2 methylation sites are unmethylated. A fourth, further reduced fluorescence signal VA4 is obtained when 3 of the methylation sites are methylated and 1 methylation site is unmethylated. Finally, a fifth, most reduced or weakest fluorescence signal VAS is obtained when all 4 methylation sites are methylated. This measure makes it possible to determine exactly how many of the potential methylation sites in the target nucleic acid molecule are methylated and how many are unmethylated.

[0044] In yet another embodiment then said method comprises the further following step: g) Comparing the multiple distinct values AI , A2, A3, VAnfor quantification of each of the specific patterns.

[0045] In this step, the values AI , A2, A3, VAnare compared with each other or set in relation to each other in order to determine which value, in relative terms, is present with which strength or size. This measure allows the quantities of the respective methylation species to be determined by comparing the signal strengths. Thisquantification is particularly simple and accurate when VAI, VA2, VA3, VAnare represented in the form of RFUs.

[0046] In an embodiment of the invention, in step (b) further a quantity detection probe is provided configured to specifically bind to non-methylatable nucleotides of said nucleic acid molecule with a binding efficiency value VQ, and, optionally, said quantity detection probe is incubated in step (c).

[0047] The "quantity detection probe" is configured so that it binds outside the 'methylation detection probe', because it does not bind to methylatable nucleotides, but it still binds inside the PCR amplicon. The methylation detection probe therefore always binds in the same way, completely independently of the methylation or the degree of methylation of the methylatable nucleotides in the target nucleic acid molecule. When performing PCR in a duplex reaction, not only the number of methylated nucleic acid molecules can be directly quantified based on the number of methylated sites covered by the methylation detection probe, but also the percentage of methylation in relation to the total amount of nucleic acid molecules in the PCR reaction can be determined.

[0048] The quantity detection probe binds to the non-methylatable nucleotides with a binding efficiency value VQ. In one embodiment of the invention, this can be easily determined if the quantity detection probe also has a detectable marker, preferably a fluorescence marker. In this case, in a further embodiment example, Q is again represented by relative fluorescence units (RFU). In a further embodiment example, the quantity detection probe is also designed as a TaqMan probe. Preferably, a different fluorescence marker is then provided on the quantity detection probe than on the methylation detection probe, e.g., FAM vs. HEX. This is an advantageous way of distinguishing between the two signals.

[0049] In another embodiment said method comprises the further following step:h) Comparing VA with Q for quantification of methylation of said nucleic acid molecule.

[0050] This measure is an advantageous way of determining the percentage or proportional amount of methylated nucleic acid molecule in the total amount of nucleic acid molecule in the sample.

[0051] In yet another embodiment of the invention the method comprises the following step: h') Comparing each of VAI , VA2, VA3, VAnwith VQ for quantification of methylation patterns of said nucleic acid molecule.

[0052] This measure can even be used to determine the percentage or proportional amount of a single methylation species in the total amount of the nucleic acid molecule in the sample. This embodiment provides a more precise quantification of methylation by considering multiple regions separately. This precision is crucial for understanding complex methylation landscapes.

[0053] Another subject-matter of the invention relates to a second method of detecting the methylation status of a plurality of nucleic acid molecules, comprising the following steps: a) Providing a plurality of first nucleic acid molecules having a first nucleotide sequence comprising methylatable nucleotides of unknown methylation status; b) Providing a plurality of second nucleic acid molecules having said first nucleotide sequence comprising said methylatable nucleotides of unmethylated status; c) Providing a methylation detection probe configured to specifically bind to said methylatable nucleotides of unmethylated status or, in the alternative,of methylated status, said methylation detection probe comprises a detectable marker activatable by polymerase chain reaction (PCR), d) Subjecting said plurality of first nucleic acid molecules together with said methylation detection probe to a first PCR, and subjecting said plurality of second nucleic acid molecules together with said methylation detection probe to a second PCR e) Determining the detectable marker of said methylation detection probe in said first PCR to obtain a first signal value Si , and determining the detectable marker of said methylation detection probe in said second PCR to obtain a second signal value S2, f) Detecting a methylation of said first nucleic acid molecule if Si < S2, or, in the alternative, if Si > S2.

[0054] The features, characteristics, advantages and embodiments described for the first method according to invention at the outset apply to said second method according to the invention correspondingly. Whenever an embodiment of the second method according to the invention is specified, explicit reference is made to the explanations of the corresponding embodiment of the first method according to the invention.

[0055] The second method comprises two alternatives. According to the first alternative, the methylation detection probe is designed such that it hybridizes perfectly to the methylatable nucleotides in the unmethylated state, in the second alternative, the methylation detection probe is designed such that it hybridizes perfectly to the methylatable nucleotides in the methylated state.

[0056] Si and S2 of the second method according to the invention relate to VA and VR of the first method according to the invention, however, the binding efficiencies are directly correlated to signal strengths.

[0057] According to an embodiment of said second method according to the invention after step (b) and before step (c) the following step is carried out:b') subjecting the nucleic acid molecule to a bisulfite conversion reaction.

[0058] According to another embodiment of said second method according to the invention, said PCR is digital PCR (dPCR).

[0059] In still another embodiment of said second method said detectable marker is a fluorescence marker.

[0060] In yet another embodiment of the second method according to the invention Si and S2 are determined by relative fluorescence units (RFU).

[0061] In a further embodiment of the second method according to the invention the methylation detection probe is configured as a TaqMan probe.

[0062] In another embodiment of the second method according to the invention Si comprises multiple distinct values (S1.1 , S1.2, S1.3, Si.n) each representing a specific pattern of methylated nucleotides comprised by said first nucleic acid molecule.

[0063] S1.1, S1.2, S1.3, Si.nrelate to VAI , VA2, VA3, VAnof the first method according to the invention, however, the binding efficiencies are directly correlated to signal strengths.

[0064] I an embodiment of the second method according to the invention in step (d) further subjecting to the first PCR and, optionally, the second PCR, a quantity detection probe configured to specifically bind to non-methylatable nucleotides of said first nucleotide sequence, said quantity detection probe comprises a detectable marker activatable by PCR.

[0065] In yet another embodiment of the second method according to the invention said detectable marker of said quantity detection probe is a fluorescence marker, and, preferably, the quantity detection probe is configured as a TaqMan probe.

[0066] In still another embodiment of the invention in step (e) further the detectable marker of said quantity detection probe in said first PCR and, optionally, said second PCR, is determined to obtain a third signal value S3.

[0067] S3 relates to VQ of the first method according to the invention.

[0068] In yet another embodiment of the second method according to the invention, S3 is represented by relative fluorescence units (RFU).

[0069] In a further embodiment the second method according to the invention comprises the further following step: i) Comparing Si with S3 for quantification of methylation of said first nucleic acid molecule.

[0070] In a further embodiment the second method according to the invention comprises the following step: i') Comparing each of S1.1 , S1.2, S1.3, Si.nwith S3 for quantification of methylation patterns of said nucleic acid molecule.

[0071] Another subject-matter of the invention relates to a kit for detecting methylated nucleotides comprised by a nucleic acid molecule and / or the methylation status of a plurality of nucleic acid molecules, comprising: a methylation detection probe configured to specifically bind to methylatable nucleotides of said nucleic acid molecule of unmethylated status, and optionally, a quantity detection probe configured to specifically bind to non- methylatable nucleotides of said nucleic acid molecule, and a manual comprising instructions for carrying out the first and / or second method according to the invention.

[0072] The properties, features, advantages and embodiments of the methods according to the invention apply equally to the kit according to the invention.

[0073] The advantage of providing a kit is that all components, reagents and information are provided together to enable error-free operation of the method according to the invention, even for untrained or semi-skilled personnel.

[0074] In the kit the methylation detection and the quantity detection probes 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.

[0075] The kit may further comprise 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 contain / s instructions on or associated with the container(s) that indicate directions for reconstitution and / or use.

[0076] In an embodiment of the kit according to the invention said methylation detection probe and, optionally, said quantity detection probe comprise(s) a detectable marker, preferably a marker activatable by PCR, further preferably a fluorescence marker.

[0077] In still another embodiment the kit according to the invention further comprises one or more PCR primers configured to allow amplification of said nucleic acid molecule(s) in a PCR.

[0078] 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 to which 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 presentdisclosure, 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.EXAMPLES

[0079] Before the present invention is further described, it is to be understood that this disclosure is not strictly limited to particular embodiments or examples 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 and examples only, and is not intended to be limiting, since the scope of the present invention will be limited only by the claims.

[0080] The invention is now further explained by means of examples resulting in additional features, characteristics and advantages of the invention. The features mentioned in the specific examples are features of the invention and may be seen as general features which are not applicable in the specific example but also in an isolated manner in the context of any embodiment of the invention.

[0081] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments or examples, 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 or example, 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 disclosureand are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0082] The invention is now further described and explained in further detail by referring to the following non-limiting examples and figures:Fig. 1 : Schematic display of the approach. The methylation detection probe (green) anneals to the methylation island containing 4 potential methylation sites. The probe annealing efficiency depends on the number of methylated sites. The more methylated sites are present the lower the binding efficiency and the lower the resulting RFU values in the dPCR reaction. Based on the relative RFU values the number of methylated sites can be inferred.Fig. 2: Methylation detection probe alone with single gBIocks as templates. Each gBIock was run in triplicate and reflects a different grade of methylation. From left to right 1 - 4 methylated CpG sites (and hence 1-4 mismatches reducing binding efficiency) and in the 5th triplicate the perfect matching wildtype sequence generating the highest RFU cluster. The last column contains a PCR reaction without a template (NTC). Red line shows the threshold set to distinguish negative and positive partitions.Fig. 3: Detection of more complex methylation patterns. To simulate a more complex methylation pattern, three gBIocks were tested in single plex and combined together and tested in triplicate reactions. The pattern forming in the mixed PCR reaction corresponds to the RFU levels of the clusters presenting in single gBIock reactions. First, meth. gBIocks one and three were combined with the non-meth. gBIock (upper panel) followed by meth. gBIocks two and four combined with the non-meth. gBIock (lower panel).Fig. 4: Elucidation of more complex methylation patterns. All five gBIocks were run alone, each PCR reaction resulting in a distinct cluster with a distinguishable RFU value. Each reaction was performed in triplicate (panels 1-5 from the left).In the rightmost panel, a complex methylation pattern was simulated by mixing all 5 gBIocks together. Each methylation state results in a distinct cluster that matches it’s single plex result in RFU values.Fig. 5: Schematic overview of the two probe approach. The quantity detection probe detects DNA regardless of methylation and always generates a signal. The methylation detection probe detects methylated CpG sites that are covered by the probe.Fig. 6: Methylation detection assay in combination with a quantity detection probe. The upper panel shows the results of the methylation detection probe. Panels 1-5 display the results of gBIocks with an increasing number of methylated sites as well as no methylation present. Panel 6 shows the mixture of all gBIocks together. The lower panel shows the results of the matching quantity detection probe for each reaction.1. Concept

[0083] The invention presented here builds on the known MethyLight approach combined, in an embodiment, with the advantages of digital PCR (dPCR). Here, the basic idea is that a methylation detection probe, in another embodiment, configured as a single TaqMan probe, designed to perfectly match the unmethylated sequence of methylatable nucleotide, such as a cytosine of a CpG island, generates a certain level of relative fluorescence when an unmethylated DNA template is being used. When a single CpG site in the template presents as methylated, the sequence changes in one position and the TaqMan probe now covers a single mismatch. This results in a reduced binding efficiency of the TaqMan probe and subsequently in a reduced level of relative fluorescence. This effect is more pronounced the more CpG sites present methylated and therefore creating mismatches in the binding of the TaqMan probe. Many reactions together then form a distinct cluster that can be identified and matched to a certain methylation status. I.e., each methylation "pattern" creates its own RFU cluster (Figure 1). Based on the pattern of the clusters the number of methylated sites can be inferred.

[0084] In addition to the number of methylated sites, the invention, in another embodiment, further uses a second quantity detection probe, in another embodiment also configured as a TaqMan probe, which is designed to anneal to a sequence beside the methylation detection probe, but within the PCR amplicon. The idea is to have a quantity detection probe that will always indicate how many total DNA templates were accessed in the PCR reaction. This allows to determine what the percentage of DNA in the reaction was methylated to which degree prior to bisulfate modification, i.e. , not only the overall grade but also on an individual level i.e. how many DNA had how many methylated CpG sites.2. Material and methodsNucleic acids, sequences

[0085] All primers, TaqMan probes and gBIocks were purchased from IDT. gBIocks were diluted down to either 1000 cp / pl reaction for single plex experiments or 200 cp / pl reaction when mixed to simulate complex methylation patterns using nuclease free water. Primers and TaqMan probes were used in concentrations recommended by Qi- agens one-step advanced probe mix handbook. All sequences used are shown in Table 1.Table 1 : Assay sequencesDigital PCR

[0086] The dPCR reactions were set up according to standard QIAcuity One- Step advanced probe PCR Kit Quick Start Protocol. The Nanoplates were sealed and placed in a QIAcuity Digital PCR instrument according to the instrument's user manual. Standard QIAcuity dPCR cycling program (the annealing step was set to 60°C and was run for 60 seconds) was run for 50 cycles. Standard gain and exposure were used for imaging. Results were analyzed using QIAcuity Software Suite (Suite 2.5). The assays were run in a triplex reaction to eliminate potential experimental bias.3. Results

[0087] The inventors designed 5 individual gBIocks encoding one of the MGMT gene methylation island (Table 1). One gBIock was designed with no methylated CpG site and the other four gBIocks with up to four methylated CpG sites, simulating different degrees of methylation. For example meth. gBIock I corresponds to the gBIock that contains a single methylated CpG site. All other gBIocks are named accordingly.

[0088] First, the inventors tested each of the 5 gBIocks individually and determined the mean RFU of the main cluster (Figure 2).

[0089] With only one mismatch (i.e. one methylated CpG site, leftmost triplicate) the RFU of the main cluster is lower than the RFU level of the non-meth gBIock (2nd triplet of plots from the right). With an increasing number of mismatches the PCR efficiency decreases further and hence the resulting RFU of the PCR reaction (Figure 2). This demonstrates the functionality of the assay system.

[0090] The use of a single gBIock simulates a situation were only one pattern of methylation is present in the specific CpG island. When a mixture of methylation patterns is present in the sample of interest the assay system reacts by displaying distinct clusters for each methylation pattern (Figure 3). The inventors simulated such a situation by mixing the non-meth. gBIock with meth.gBlockl and three. The pattern emerging on the rightmost triplet of Figure 3 (upper panel) demonstrates the ability of the assay system to distinguish between different methylation patterns. The RFU values of the individual clusters correspond to the gBIocks run in a singular reaction (left three panels). The same can be observed when combining methgBlock2 with gBlock4 and the matching non-meth. gBIock (Figure 3, lower panel).

[0091] The approach described here can also resolve more complex methylation patterns beyond the two methylated versions shown in Figure 3. Increasing the number of methylation sites from two to four in addition to the wildtype gBIock, results in a distinct 5 cluster pattern were each gBIock (and hence methylation state) refers to anindividual cluster at the same RFU levels as the gBIock alone. Figure 4 illustrates the capacity of the assay system presented here to resolve 5 different patterns. The ability to design a TaqMan probe across several possible methylation sites leads to more flexibility in assay design.

[0092] Furthermore, the idea was to extent the approach to be capable to determine the contribution of each methylated cluster to the total amount of DNA added to the PCR reaction.

[0093] The assay system was therefore expanded to incorporate a second TaqMan Probe (HEX labelled), named quantity detection probe, which anneals in a position outside of the methylated TaqMan probe but still within the PCR amplicon (Figure 5). When both assays are being run in a duplex reaction, not only the number of methylated DNA molecules can be directly quantified based on the number of methylated CpG sites covered by the methylation probe, but also the percentage of methylation in regards to the total amount DNA present in the PCR reaction (Figure 6).

[0094] As displayed in Figure 6, the upper panel show the results of the methylation detection probe. In the gBIock 1-5 panel, each cluster corresponds to a different methylation status. Matching the different clusters to a gBIock is possible by comparing the RFU values of each cluster to the ones of the individual gBIocks (panel meth. gBIock 1-4 plus non-meth). Each cluster can then be directly quantified. The lower panel of Figure 6 show the results of the quantity detection probe, detecting all the DNA templates in the PCR reaction. Both quantification results together determine what percentage of DNA templates were methylated overall. When calculated vs. each cluster (upper panel, gBIock 1-5) the approach can even determine what percentage of total DNA was methylated to which degree. Due to the fact that the control runs within the same amplicon, there is no need for additional controls or secondary PCR reactions.quantification results in copies / pl reaction. On the left panel the results of the methylation detection probe and on the right panel the results of the quantity detection probe in the same amplicon are displayed.

[0095] The absolute quantification results of both probes in the duplex reaction (Table 2) show a near identical quantification. Meaning 100% of the DNA added to the reaction was detected by both probes. It further shows, that imperfect annealing of the methylation probe is not impairing the quantification of templates.

[0096] Finally, when delineating the complex methylation pattern down to the individual gBIocks each can be individually quantified and compared to the quantification results of the second quantity detection probe. As expected from preparing the gBIock mix, each probe made up -20% of the total amount.

[0097] Taken together, here the inventors describe an assay system that is capable of delineate and quantify complex methylation patterns with a higher degree of design freedom and without the need for additional controls or secondary standards.

Claims

Claims1. A method of detecting methylated nucleotides comprised by a nucleic acid molecule comprising the following steps: a) Providing a nucleic acid molecule suspected of comprising methylated nucleotides; b) Providing a methylation detection probe configured to specifically bind to methylatable nucleotides of said nucleic acid molecule, said specific binding to said methylatable nucleotides in non-methylated state occurs with a reference binding efficiency value VR; c) Incubating said nucleic acid molecule and said methylation detection probe under conditions allowing the specific binding of said methylation detection probe to said nucleic acid molecule, and d) Determining a value VA of an actual binding efficiency of the methylation detection probe to the nucleic acid molecule, e) Comparing VA and R, and f) Detecting methylated nucleotides if VA < VR.

2. The method of claim 1, wherein after step (a) and before step (b) the following step is carried out: a') subjecting the nucleic acid molecule to a bisulfite conversion reaction.

3. The method of claim 1 or 2, wherein in step (c) said nucleic acid molecule and said methylation detection probe are subjected to a polymerase chain reaction (PCR), preferably digital PCR (dPCR).

4. The method according to any one of the preceding claims, wherein the methylation detection probe comprises a detectable marker configured to allow determination of VA, preferably a fluorescence marker.

5. The method of any of the proceeding claims, wherein the methylation detection probe is configured as a TaqMan probe.

6. The method of any of the proceeding claims, wherein VA comprises multiple distinct values ( AI, VA2, VA3, VAn) each representing a specific pattern of methylated nucleotides comprised by said nucleic acid molecule.

7. The method of claim 6, comprising the further following step: g) Comparing the multiple distinct values AI, VA2, VA3, VAnfor quantification of each of the specific patterns.

8. The method of any of claims 3 to 7, wherein in step (b) further a quantity detection probe is provided configured to specifically bind to non-methylatable nucleotides of said nucleic acid molecule with a binding efficiency value Q, and, optionally, said quantity detection probe is incubated in step (c).

9. The method of claim 8, wherein the quantity detection probe comprises a detectable marker configured to allow determination of Q, preferably a fluorescence marker.

10. The method of claims 8 or 9, wherein the quantification probe is configured as a TaqMan probe.

11. The method of any of claims 8 to 10, comprising the further following step: h) Comparing VA with VQ for quantification of methylation of said nucleic acid molecule.

12. The method of claim 11 , comprising the following step: h') Comparing each of VAI , VA2, VA3, VAnwith VQ for quantification of methylation patterns of said nucleic acid molecule.

13. A method of detecting the methylation status of a plurality of nucleic acid molecules, comprising the following steps: g) Providing a plurality of first nucleic acid molecules having a first nucleotide sequence comprising methylatable nucleotides of unknown methylation status; h) Providing a plurality of second nucleic acid molecules having said first nucleotide sequence comprising said methylatable nucleotides of unmethylated status; i) Providing a methylation detection probe configured to specifically bind to said methylatable nucleotides of unmethylated status or, in the alternative, of methylated status, said methylation detection probe comprises a detectable marker activatable by polymerase chain reaction (PCR), j) Subjecting said plurality of first nucleic acid molecules together with said methylation detection probe to a first PCR, preferably a digital PCR (dPCR), and subjecting said plurality of second nucleic acid molecules together with said methylation detection probe to a second PCR, preferably a dPCR, k) Determining the detectable marker of said methylation detection probe in said first PCR to obtain a first signal value Si , and determining the detectable marker of said methylation detection probe in said second PCR to obtain a second signal value S2, l) Detecting a methylation of said first nucleic acid molecule if Si < S2, or, in the alternative, if Si > S2.

14. The method of claim 13, wherein after step (b) and before step (c) the following step is carried out:b') subjecting the nucleic acid molecule to a bisulfite conversion reaction.

15. A kit for detecting methylated nucleotides comprised by a nucleic acid molecule and / or the methylation status of a plurality of nucleic acid molecules, comprising: a methylation detection probe configured to specifically bind to methylatable nucleotides of said nucleic acid molecule of unmethylated status, and optionally, a quantity detection probe configured to specifically bind to non- methylatable nucleotides of said nucleic acid molecule, and a manual comprising instructions for carrying out the method of any of claims 1 to 14.

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