[0001] The present invention relates to the means and methods for the detection of the methylation status of cytosine residues and in particular to the use of at least one primer which is at least 5 nucleotides in length, and which hybridizes to a target sequence that comprises at least one defined 5 ^' -CpG-3 ^' which target sequence is comprised in a genomic DNA of a vertebrate, and which has its 3 ^" end opposite to the C of said 5 ^' -CpG-3 ^' , and which comprises at said 3 ^" end a mismatched nucleotide in relation to said C of said 5 ^' -CpG-3 ^' , for determining the methylation status of a vertebrate genomic nucleic acid at said C. The present invention further relates to polymerase proteins that can be used in the embodiments of the present invention. The present invention also relates to a kit comprising at least one primer of the invention and optionally means to conduct the amplification. The present invention further relates to a method for directly detecting methylation or hydroxymethylation of a cytosine residue of interest in a DNA molecule. Said method comprises steps of providing a primer having its 3'-end opposite of the cytosine residue of interest and having at said 3'-end a mismatched base, performing a specific DNA polymerase reaction, such as primer extension, rolling circle amplification (RCA) or polymerase chain reaction (PCR), with said primer using said DNA molecule as template, and detecting said methylation or hydroxymethylation via an increased efficiency of said specific DNA polymerase reaction as compared to the same reaction performed with an unmodified DNA molecule as template.

[0002] Different cells of an organism display broad functional and morphological diversity, although they all possess the same genetic material. Differential gene expression is the cause for this heterogeneity. The term "epigenetics" relates to all research in this field. It is defined as the study of inheritable, phenotypical changes in the gene expression pattern of a specific cell type that are not caused by a transformed nucleotide sequence of the genetic code itself. A coding for the gene expression state, which was postulated for the first time 36 years ago, is flexible enough to support specialization of genetically identical somatic cells towards different functions and to enable reactions to regulatory impacts from other cells or from external stimuli. Further, this coding is stable enough to persist in the germ cells and to be passed from one generation to the next. Epigenetic markers are represented by a variety of molecular mechanisms, such as posttranslational histone modifications, ATP-dependent chromatin remodeling, small and other non-coding RNA (siRNA, miRNA), binding of histone variants and non-histone proteins, polycomb-trithorax protein complexes and last but not least DNA methylation and hydroxymethylation.

[0003] 5-Hydroxymethylcytosine (Fig. 1 C) was first discovered in the bacteriophages T2, T4 and T6 in 1952. The presence of it in mammalian DNA was suggested not until twenty years later, but has received only little scientific attention. In 2009, 5-hydroxymethylcytosine was detected in cerebellar Purkinje neurons in the brain, where it constitutes 0.6% and 0.2% of all bases in Purkinje cells and granule cells, respectively. Simultaneously, 5- hydroxymethylcytosine was reported to be present in mouse embryonic stem cells and human embryonic kidney cells. The TET1 (ten-eleven translocation 1 ) protein, a fusion partner of histone methyltransferase in acute myeloid leukemia, was identified as a 2-oxoglutarate- and Fe(l Independent enzyme that catalyzes the conversion of 5-methylcytosine to 5- hydroxymethylcytosine in vitro, as well as in cultured cells. The three paralogous human proteins TET1 , TET2 and TET3 were found as they have homologous regions to the oxygenase domains of JBP1 and JBP2 that are known to catalyze the initial step of base J (β- D-glucosyl hydroxymethyluracil) biosynthesis in trypanosomes. This is a modified thymine that is associated with gene silencing like 5-methylcytosine, as it is described herein. Moreover, it has been shown that prokaryotic cytosine-5 methyltransferases were able to produce 5- hydroxymethylcytosine by reversible addition of formaldehyde to cytosine. It was supposed that 5-hydroxymethylcytosine is formed at 5-methylcytosine sites in response to oxidative stress. For example, a study showed that 5-methylcytosine yields in 5-hydroxymethylcytosine under Fenton conditions (Fe2+, Cu2+ and H202). It is speculated that 5- hydroxymethylcytosine together with 5-formyldeoxycytosine could be one of the main oxidative degradation products of 5-methylcytosine. However, the oxidative formation of 5- hydroxymethylcytosine in vivo could not yet be confirmed. Another speculation is that 5- hydroxymethylcytosine could be an intermediate in the pathway of an active demethylation, as active methylation has been observed during different steps of development. However, the responsible enzymes have been elusive. Recently, two studies showed that 5-methylcytosine as well as 5-hydroxymethylcytosine are oxidized to 5-formylcytosine and 5-carboxylcytosine by Tet dioxygenases in cultured cells and in vitro, and that thymine-DNA glycosylases specifically recognize and excise 5-carboxylcytosine as a part of base excision repair. Additionally, it was shown by immunostaining of mitotic chromosomes that 5-hydroxymethylcytosine in the paternal genome is gradually lost during preimplantation development. It was suggested that this is a DNA-replication-dependent passive process. [0004] Methylation of cytosines at the C5-atom (Fig. 1 B) is the most abundant DNA modification in vertebrates. 5-Methylcytosine is an important epigenetic marker and plays a crucial role for activating or silencing genes. The dynamic changes of DNA methylation patterns are very important for the development of vertebrates, e.g. they are responsible for X- inactivation, imprinting and the development of primordial germ cells. Furthermore, DNA methylation plays a crucial role in several diseases such as cancer.

[0005] Several methods for the detection of DNA methylation are known in the art. The most common method is bisulphite sequencing. It is based on the selective chemical deamination of unmethylated cytosine to uracil by sodium bisulphite. Thereby, the modified cytosine analogue 5-methylcytosine remains unchanged and shows up as cytosine in the subsequent sequencing. This method can give information about all 5-methylcytosine positions in a DNA sample. Bisulphite sequencing was primarily developed to explore secondary structures of DNA. Hereby, it was discovered that the modification of cytosines through bisulphite is only then efficient, if the DNA sample is available in single strands. The first step in bisulphite sequencing is therefore the denaturation of DNA by NaOH. As the actual bisulphite reaction is carried out in slightly acidic environment, care has to be taken that the single strands do not renaturate after neutralization. This is achieved by embedding the DNA single strands in agarose beads immediately after denaturation. Bisulphite induces the conversion of cytosine to uracil. The DNA is incubated in 3 M NaHS0 ₃ and hydrochinone for several hours at 50°C. Hereby, a slow sulphonation at position C6 of the cytosine takes place. All 5-methylated cytosines are inert against this reaction. Hydrochinone is added to the reaction to inhibit the oxidation of the cytosine sulphonates with aerial oxygen. 6-Cytosine-sulphonate is spontaneously deaminated in aqueous solution. Ammonium is formed as a by-product. Then, NaOH, which is added again, leads to cleavage of uracil sulphonate into uracil and bisulphite. After the conversion reaction by bisulphite, the DNA sample is amplified by PCR. Two strategies are possible. First, two primer pairs are chosen which span the 5 ^' -CpG-3 ^' site. Hereby, one primer pair is designed for unmethylated DNA and the other primer pair for methylated DNA. This is called methylation specific PCR. Second, only one primer pair is used which flanks a 5 ^' -CpG-3 ^' site. During DNA synthesis, each 5-methylated cytosine is replaced by an unmethylated cytosine and each uracil is replaced by thymine. 5-Hydroxymethylcytosine reacts with bisulphite to yield cytosine-5-methylenesulfonate which does not promote deamination and therefore, also codes as cytosine. As a result, sodium bisulphite treatment does not distinguish between 5-methylcytosine and 5-hydroxymethylcytosine. [0006] Using this method, genome-wide methylation maps across different cell types and in response to several environmental influences were established. Various examples of methylation maps are available, e.g. for fibroblasts and embryonic stem cells in the human genome, for the Arabidopsis thaliana genome, and for a mouse genome. However, bisulphite sequencing has many disadvantages. Bisulphite sequencing uses very harsh chemicals and can cause DNA fragmentation. Due to the bisulphite conversion, the sequence, if unmethylated, is reduced to only three nucleotides (A, G, T(U)). This complicates the primer design and alignments to the reference sequence. Furthermore, two types of bisulphite conversion errors can occur: either an inappropriate conversion of 5-methylcytosine to thymine or the failure to convert unmethylated cytosine to uracil. The frequency of the error that is mentioned first was found to range from 0.09 to 6.1 % for selected protocols. The frequency of the second mentioned error is more difficult to estimate. It was proposed that the conversion rate for one cytosine is dependent on the sequence context. Lastly, as mentioned above, bisulphite sequencing cannot be used for discrimination between 5-methylcytosine and 5- hydroxymethylcytosine.

[0007] Further methods for the analysis of DNA methylation include restriction analysis of genomic DNA using methylation-sensitive restriction endonucleases, the specific enzymatic labeling of 5-hydroxymethylcytosine followed by a methylation-sensitive restriction analysis, and single molecule real-time sequencing, wherein DNA polymerase kinetics can be monitored in real-time. However, all of these methods suffer from more or less severe drawbacks such as limited usability, limited accuracy, and high time-, cost- and labor-intensities.

[0008] Accordingly, the technical problem underlying the present invention is to provide means and methods for the detection of the methylation status of a DNA molecule.

[0009] The solution to the above technical problem is achieved by the embodiments characterized herein and in the claims.

[0010] The detection of the methylation status is, as mentioned hereinbefore, a promising and emerging tool for diagnostics, prognostics, and prediction of response to therapies that awaits broad application in the future for cancer and other diseases such as neuro-developmental and metabolic disorders as well as auto-immune diseases. However, current analytical procedures prohibit broad clinical applications, because all cost-efficient and practicable methods with single nucleotide resolution that were known up to the filing date of the present application rely on bisulfite-mediated conversion of DNA. These drawbacks have moved the inventors to re-design the underlying detection methodology which ended up in the present pioneering methodology which is in essence characterized by the use of the 3 ^" -mismatched primers described herein.

[0011] The primer provided by the present invention is preferably designed such that its outmost 3'-end is opposite to the cytosine residue of interest (i.e. the one whose methylation status should be analyzed) and has a mismatched base in respect to the cytosine of interest. Said mismatched base at the 3'-end of the primer does not sufficiently pair with the cytosine of interest, in case said cytosine is not methylated or hydroxymethylated, thus impairing for example a DNA polymerase reaction with said primer using said DNA molecule as template. However, said mismatched base at the 3'-end of the primer pairs sufficiently with the cytosine of interest, in case said cytosine is methylated or hydroxymethylated, thus allowing a DNA polymerase reaction with said primer using said DNA molecule as template (see for example Fig. 2 for further illustration). The readout of said method is straightforward as it is for example merely necessary to analyze the influence of the methylation status at the cytosine residue of interest on the downstream extension reaction (see the appended examples and Figures 3 and 4 for illustration). The uses and methods of the present invention are significantly less time-, labor- and cost-intensive compared to methods known in the art. Moreover, they are much less prone to errors and allow the analysis of very small amounts of sample material.

[0012] The present invention thus relates in essence to the use of at least one primer:

(a) which primer is at least 5 nucleotides in length, and

(b) which primer hybridizes to a target sequence that comprises at least one defined 5 ^' - CpG-3 ^' which target sequence is comprised in a genomic DNA of a vertebrate, and

(c) which primer has its 3 ^" end opposite to the C of said 5 ^' -CpG-3 ^' , and

(d) which primer comprises at said 3 ^" end a mismatched nucleotide in relation to said C of said 5 ^' -CpG-3 ^' ,

for determining the methylation status of a vertebrate genomic nucleic acid at said C of said at least one defined 5 ^' -CpG-3 ^' . It is envisaged that the above embodiment makes use of the vertebrate genomic nucleic acid which comprises the target sequence as a template together with said primer.

[0013] A "primer" is an oligonucleotide, typically between about 5 to 200 nucleotides in length, capable of selectively binding to a specified nucleic acid (the target sequence) by hybridizing with the target sequence. The primer of the invention are generally made of less than 1 ,000 nucleotide (nt), including those in a size range having a lower limit of about 5 nt and an upper limit of about 500 to 900 nt. Preferred primer are in a size range having a 5 to 15 nt lower limit and a 50 to 500 nt upper limit, and particularly preferred embodiments are in a size range having a 10 to 20 nt lower limit and a 25 to 150 nt upper limit.

The term "primer" and "oligonucleotide" are used interchangeably herein. The term "oligonucleotide" includes DNA and/or RNA or modifications thereof, comprising nucleotides such as e.g. the conventional bases (A, G, C, T, U) and/or nucleotide analogs as monomeric units. An "analog" or "nucleotide analog" (used interchangeably herein) can refer to a nucleotide-like molecule such as a structural moiety that can act substantially like a nucleotide, for example exhibiting base complementarity with one or more of the bases that occur in DNA or RNA and/or being capable of base-complementary incorporation via an enzymatic reaction (e.g. via a DNA-Polymerase). "Nucleotide analogs" comprise in one example nitrogenous heterocyclic bases (e.g., inosin). The above mentioned monomeric units are typically covalently linked by standard phosphodiester bonds or other linkages. In an oligonucleotide of the invention, the backbone may be made up of a variety of linkages, including one or more of sugar-phosphodiester linkages, peptide-nucleic acid (PNA) linkages, phosphorothioate linkages, methylphosphonate linkages, or combinations thereof to name some. Locked Nucleic Acid (LNA™) nucleosides analogues are also envisaged.

The primers of the present invention can be modified and/or labeled, i.e. they may further include non-nucleotide groups such as, for example, abasic nucleotides, universal bases (e.g., 3-nitropyrrole and 5-nitroindole), polysaccharides, peptides, polypeptides etc.

By "label" is meant a reporter moiety associated with a primer which can be detected by means well known in the art and used to indicate the presence or absence of a particular polynucleotide sequence in a test sample. Examples of labels which are well known in the art include chemiluminescent, electrochemiluminescent and fluorescent compounds, radioisotopes, dyes, polynucleotides, enzymes, enzyme substrates, chromophores and haptens etc.. When multiple interacting labels are associated with a polynucleotide, interacting labels may include, for example, the following: luminescent and quencher labels, luminescent and adduct labels, dye dimer labels, enzyme and substrate labels, enzyme and cofactor labels, and Forrester energy transfer pairs. Radioactive P end-labeled primer (particularily 5 ^" -

P end labeled primer) are less preferred. In one embodiment, one, two, three, or four types of nucleotides are differentially labeled. In one such embodiment, four different types of nucleotides are labeled with four different labels. The primer of the invention may also include a "tag" sequence, which may be used to identify the primer, for example, to distinguish the primer from other, similar primer. Any nucleic acid analog is contemplated by the primers of the present invention, provided the primer can form a stable hybrid with the target sequence under hybridization conditions and, provided the primer can be extended from its 3 ^' -end. "Hybridizing" refers to the ability to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. By "hybridization conditions" or "hybridizing" or "hybridize" or grammatical variants thereof are meant conditions permitting a primer to stably hybridize to a target sequence (the complex is then e.g. stabilized via hydrogen bonding between the bases of the nucleotide residues) which is a prerequisite for initiation of template-directed synthesis of a nucleic acid complementary to the target sequence. Hybridization conditions may vary depending upon factors including the GC (guanine/cytosine) content and length of the primer, the degree of similarity between the primer sequence and sequences of non-target nucleic acids which may be present in the test sample, and the target sequence. Hybridization conditions include the temperature and composition of the hybridization reagents or solutions. Acceptable conditions could be easily ascertained by someone having ordinary skill in the art. Typically, the hybridization is performed under conditions in which the target sequence to be probed is single-stranded. The primer of the present invention has its 3 ^" end opposite to the C of said 5 ^' -CpG-3 ^' (see Fig. 2B or Fig. 3A).

The primer of the invention also provides a point of initiation for polymerase-mediated template-directed synthesis of a nucleic acid complementary to the template sequence. The "template sequence" is usually directly downstream to the target sequence. "Template" refers to a single-stranded nucleic acid, or a denatured region of a double-stranded nucleic acid, that a polymerase can utilize to synthesize a complementary nucleic acid strand.

A primer is thus capable of being extended from its 3 ^" -end, i.e. the 3'-end of the primer provides a free 3'-OH group whereto further "nucleotides" may be attached by a template- dependent DNA polymerase. "Being extended" or "extended from its 3 ^" -end" or "extending ...from its 3 ^" -end" etc. thereby includes typical "nucleic acid amplification" -methodology in all variations. Typically, nucleic acid amplification uses one or more nucleic acid polymerase and/or transcriptase enzymes to produce multiple copies of a target sequence or fragments thereof, and/or of a sequence complementary to the target sequence or fragments thereof. In vitro nucleic acid amplification techniques are well known and include transcription-associated amplification methods, such as transcription mediated amplification (TMA) or nucleic acid sequence based amplification (NASBA), and other methods such as the Polymerase Chain Reaction (PCR), quantitative real-time PCR (qRT-PCR), reverse transcriptase-PCR, replicase mediated amplification, and the Ligase Chain Reaction (LCR), to name some. A person of ordinary skill in the art will understand that other extension or amplification methods based on polymerase mediated extension of oligonucleotide sequences may be used with the compositions, uses and/or methods of the present invention.

The primer of the present invention comprises at its 3 ^" end a mismatched nucleotide in relation to said C of said 5 ^' -CpG-3 ^' . As exemplified and documented in the appended examples, the present inventors discovered that the methylation or hydroxymethylation of the "C" of said at least one defined 5 ^' -CpG-3 ^' influences the extension reaction from said 3 ^' -end when using the primer of the present invention. The rationale is as follows, without being bound by theory. The primer of the present invention has its 3 ^" end opposite to the C of said 5 ^' -CpG-3 ^' , and comprises at said 3 ^" end a mismatched nucleotide in relation to said C of said 5 ^' -CpG-3 ^' . A "mismatched" nucleotide or base (or nucleoside etc.) in relation to said "C" thereby means in essence that the very mismatched nucleotide etc. is not complementary to said "C" thereby excluding in a preferred embodiment a "G" at the utmost 3 ^' -end of the primer. It is thus preferred that the mismatched nucleotide etc. is selected from the group consisting of adenine, cytosine, thymine, inosine, uracil (A, C, T, I, U) or modifications thereof. It is envisaged that said mismatched nucleotide etc. at the utmost 3'-end of the primer does not canonically pair with the cytosine of interest, in case said cytosine is not methylated or hydroxymethylated, which negatively impairs the 3 ^' -extension reaction of said primer. The methylation/hydroxymethylation of said "C" somehow compensates for said mismatch which positively impairs the 3 ^' -extension reaction of said primer (see for example Fig. 2 for further illustration). The methylation/hydroxymethylation status of the "C" in the at least one 5 ^' -CpG-3 ^' thus influences the extension reaction from the 3 ^" -end of the primer of the invention.

[0014] Primers hybridizing to opposing strands of a double- stranded target sequence are referred to as forward and reverse primers.

[0015] In a preferred embodiment, the primer is substantially complementary to its target sequence. As used herein, "substantially complementary" in reference to two nucleic acids, means that the two nucleic acids each contain hybridization regions that are of sufficiently complementary as to be able to interact with each other in a specific, determinable fashion, i.e., when the two nucleic acids are brought together in an antiparallel orientation, the same nucleotides of each nucleic acid will become hybridized to each other at one or more specific locations. "Substantially complementary" also means that the primer is almost completely or partially complementary to its target sequence. Partially complementary encompasses primer that are at least 70%, 75%, 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 96, 97, 98 or 99% complementary to their target sequence. For the determination of sequence identity, a comparison is made by aligning the sequences in a manner to provide the maximum correspondence of nucleotides. It is envisaged, however, that the primers of the present invention are in a preferred embodiment not 100% complementary to their target sequence because at least the 3 ^" -end of the primer of the invention is mismatched in relation to the C of the 5 ^' -CpG-3 ^' , as described herein elsewhere. In some embodiments, the two hybridization regions may have a maximum of 30 mismatches, 20 mismatches, 10 mismatches, or 7 mismatches. In still other cases, the two hybridization regions may have a maximum of 6, 5, 4, 3, 2, or 1 , mismatches.

[0016] In a particularly preferred embodiment of the present invention, the primer further comprises the base C (e.g. contained in a nucleotide or nucleoside) at the penultimate position in relation to its 3 ^" end.

[0017] Determining the methylation status of a vertebrate genomic nucleic acid at said "C" means the "C" that is comprised in a 5 ^' -CpG-3 ^' site. The "methylation status" thereby denotes the presence or absence of a methyl-residue or hydroxymethyl-residue at said "C", and in particular at the C5-atom of said "C" (see Fig. 1 for illustration). It is envisaged that "determining the methylation status" comprises a qualitative detection (presence or absence) and/or a quantitative or semi-quantitative detection of the methylation/hydroxymethylation status. The terms "determining," "measuring," "evaluating," "assessing," and "assaying" are used interchangeably herein to refer to any form of measurement, and include determining if an element is present or not. These terms include both quantitative and/or qualitative determinations. Assessing may be relative or absolute.

The methylation status is also exemplified herein elsewhere (for example in the following paragraph).

Cytosine methylation is crucial for mammalian embryogenesis. During this process, methylation levels change dynamically. There are various cell-type specific epigenomes with a well-defined methylation pattern which occurs in differentiation of the mammalian organism. Differentiation is characterized by two waves of genome-wide epigenetic reprogramming in the zygote and in the primordial germ cells. The genome becomes demethylated during preimplantation in mice. The maternal genome remains methylated or undergoes de novo methylation, whereas the paternal genome is rapidly and actively demethylated. Through cell divisions, the loss of maternal methylation markers occurs passively until blastocyst formation. In implementation, when the cell lines start to develop to different lineages, the methylation level is restored de novo. All DNA methyltransferases, which are responsible for the methylation of cytosines, are essential and a dysfunction in any of them leads to embryonic lethality. The second wave only occurs in the primordial germ cells where DNA methylation patterns are deleted at all single-copy genes. Ageing and cellular senescence are also characterized by a decrease of the overall content of DNA methylations. However, specific sites of distinct genes acquire methylation, for example at their promoters. This situation is similar to methylation changes in cancer or other diseases.

DNA methylation of the "C" in 5 ^' -CpG-3 ^' within or in proximity to genetic elements such as promoters regulates the transcription of the corresponding genes. A hypomethylated promoter leads to active gene expression, whereas a gene with a hypermethylated promoter is silenced. It is supposed that 5 ^' -CpG-3 ^' methylation directly disturbs the binding of transcriptional regulators to their appropriate DNA sequences. Another possibility could be the recruitment of methyl-5 ^' -CpG-3 ^' binding proteins which leads to a repressed chromatin environment. Furthermore, DNA methylation is closely interconnected with chromatin remodeling and histone modification. It is a system of multiple layers of epigenetic modifications to modulate gene expression through chromatin structure, as transcription does not act on naked DNA, but on chromatin, which is responsible for the DNA accessibility to transcription factors. However, an unmethylated state of a CpG island does not always correlate with a transcriptional active gene. The gene can be potentially activated. On the other hand, silencing of genes is not necessarily induced by the simple presence of methylation. A specific promoter core region which spans the transcription start is often, but not always, crucial for gene expression. Thus, methylation of specific 5 ^' -CpG-3 ^' sites might correlate better with gene expression than the methylation state of the whole CpG island.

In somatic cells, about 1 % of DNA bases are 5-methylcytosines. The abundance of 5- methylcytosine varies slightly in different tissue types. 5-Methylcytosines are frequently found as symmetrical 5-methylations of the dinucleotide CpG within or nearby promoters. Here, 75% of them are methylated throughout the mammalian genomes. CpG dinucleotides are underrepresented in the genome since they are mutation hotspots. Methylated CpGs can be deaminated to the naturally occurring DNA bases TpGs which cannot be repaired. Therefore, mutation rates of CpG sites are about 10 to 50 times higher than other transitional mutations and have led to depletion of the dinucleotide during evolution. However, CpG-rich clusters of a length of one to four kilobases are observed in promoter regions and the first exon of various genes. They are called CpG islands of which there are about 30,000 in the human genome. CpG islands typically occur at or near the transcription start site of genes, particularly housekeeping genes, in vertebrates. 88% of active promoters are associated with CpG-rich sequences and might be regulated by DNA methylation. Their susceptibility to become methylated alters during development and carcinogenesis. [0018] It is also envisaged that the embodiments according to the invention are used in diagnostics, for diagnostic analysis or for bioanalytics, or for the screening of tissue or fluids from the human or even animal body for the presence of certain methylation pattern. Other possible uses are disclosed herein elsewhere.

[0019] The term "target sequence" denotes said part of vertebrate genomic nucleic acid that comprises at least one defined 5 ^' -CpG-3 ^' which is the 5 ^' -CpG-3 ^' of interest and represents the sequence to which the primer hybridizes to.

The target sequence is or is comprised in a vertebrate genomic nucleic acid. Said vertebrate genomic nucleic acid/DNA or "genomic nucleic acid/DNA of a vertebrate" is either isolated (for example extracted from a vertebrate by standard methods) or still part of an isolated biological sample of said vertebrate (e.g. a cell sample, such as a blood and/or cancer cell etc.; a body fluid, such as blood, serum, liquor, cerebrospinal fluid, amniotic fluid, peritoneal fluid, interstitial fluid, excretions (urine, stool) etc.; and/or or tissue such as biopsy material, cancer tissue etc.). The source of the tissue or cell sample may be solid tissue as from a fresh, frozen and/or preserved organ or tissue sample or biopsy or aspirate. The tissue sample may also be primary or cultured cells or cell lines. Optionally, the tissue or cell sample is obtained from a disease tissue/organ. In a preferred embodiment, said vertebrate genomic nucleic acid is a mammalian genomic nucleic acid. In a more preferred embodiment, said mammalian genomic nucleic acid is human genomic nucleic acid (including mitochondrial DNA). The vertebrate genomic DNA may comprise the whole genome or only parts thereof (such as single chromosomes, mitochondrial DNA), or fragments thereof (e.g. fragments of a particular size range, generally of about 200-600 base pairs). It is also envisaged that the vertebrate genomic DNA is physically fragmented for example sonicated, sheared, or enzymatically fragmented etc. It is further envisaged that the vertebrate genomic DNA is at least 35nt in length. It is also envisaged that the genomic DNA is single or double stranded.

[0020] It is also envisaged that the genomic DNA (in particular the fragments) are immobilized to a solid support. Using the embodiments of the present invention, multiple genomic DNA fragments may be separated and analyzed by immobilizing the genomic DNA fragments on discrete areas of a solid support, e.g., on a microarray. In this manner, genomic DNA fragments of interest may be immobilized and anaylzed in a high throughput manner. The solid support can have a variety of configurations, e.g., including, but not limited to, planar supports, non-planar supports, a sheet, bead, particle, slide, wafer, web, fiber, tube, capillary, microfluidic channel or reservoir, or other structure. The solid support may be porous or non- porous. The substrate may be formed from any suitable material, depending upon the application. For example, the substrate may be a silicon-based chip or a glass slide. Other suitable substrate materials for the arrays of the present invention include, but are not limited to, glasses, ceramics, plastics, metals, alloys, carbon, agarose, silica, quartz, cellulose, polyacrylamide, polyamide, polyimide, and gelatin, as well as other polymer supports or other solid-material supports. A "microarray," includes any one-dimensional, two-dimensional or substantially two-dimensional (as well as a three-dimensional) arrangement of addressable regions bearing a particular chemical moiety or moieties.

[0021] It will be understood that the vertebrate genomic nucleic acid comprises the target sequence and the template sequence that is employed in the context of the present invention.

[0022] The target sequence of the present invention comprises at least one defined 5 ^' -CpG-3 ^' (sometimes also denoted as CpG site or 5 ^' -CpG-3 ^' or the like). "CpG" is shorthand for "— C— phosphate— G— ", that is, cytosine and guanine separated by only one phosphate. Said at least one 5 ^' -CpG-3 ^' can be comprised by a CpG island. A CpG island is usually a region comprised in a genomic DNA of a vertebrate, with at least 200 bp, and a GC percentage that is greater than 50%, and with an observed-to-expected CpG ratio that is greater than 60% (see for example Gardiner-Garden et al., Journal of Molecular Biology 196 (2): 261-82.). The "observed-to-expected CpG ratio" is calculated by formula ((Num of CpG/(Num of C ^χ Num of G)) x Total number of nucleotides in the sequence) "Defined 5 ^' -CpG-3"' means the 5 ^' -CpG- 3 ^' of interest. In a preferred embodiment, said target sequence is comprised by a gene depicted in the Tables herein (including the Table in Figure 5).

[0023] In a preferred embodiment, said at least one defined 5 ^' -CpG-3 ^' , or the target sequence, is comprised in a genetic element. The term "genetic element" includes for example regulatory elements such as a promoter, enhancer, silencer, insulator etc.; a gene or a part thereof such as an intron or exon; an intron/exon boundary, and/or a splicing region or other well-known genetic elements that can be identified in a genome of a vertebrate, such as transposons, etc.

[0024] It is further envisaged that said at least one defined 5 ^' -CpG-3 ^' , or the target sequence, is comprised in a vertebrate genomic nucleic acid or fragment thereof and/or in a genetic element, wherein said vertebrate genomic nucleic acid or fragment thereof and/or said genetic element is associated with a disease (or disease state), such as for example a tumor suppressor gene, a genetic element controlling the expression of a tumor associated antigen etc. (exemplified herein). The diseases mentioned include but are not limited to neurological diseases, metabolic diseases, cardiovascular diseases, autoimmune diseases, cancer etc. nucleic acid molecules It is also envisaged that said at least one defined 5 ^' -CpG-3 ^' is comprised in a vertebrate genomic nucleic acid or fragment thereof and/or in a genetic element, that is or is assumed to be differentially methylated in a disease. Such genetic elements (or fragments of a vertebrate genomic nucleic acid) are exemplified herein. A preferred selection comprises BRCA1 , MGMT PYCARD, RASSF1A, SHOX2 and/or SEPT9 but is not limited thereto.

[0025] In a further preferred embodiment of the methods, uses and kits of the present invention, said vertebrate genomic nucleic acid, and/or the target sequence, is not bisulfite treated. "Not bisulfite treated" means in essence that at least the target sequence that is employed in the context of the present invention, preferably the vertebrate genomic nucleic acid that is employed in the context of the present invention, is not suitable for/not prepared for bisulfite sequencing. "Bisulfite treated" or "Bisulfite treated" likewise refers to exposure of a nucleic acid to bisulfite ion (e.g., magnesium bisulfite or sodium bisulfite) at a concentration sufficient to convert unprotected cytosines to uracils. "Bisulfite treatment" also refers to exposure of a nucleic acid to other reagents that can be used to convert unprotected cytosines to uracils, e.g., disulfite and hydrogensulfite, at an appropriate concentration. "Bisulfite treatment" generally includes exposure of the nucleic acid to a base, e.g., NaOH, after exposure to the bisulfite ion or other reagent. Bisulfite sequencing is characterized by the selective chemical deamination of unmethylated cytosine to uracil by sodium bisulfite, as explained herein elsewhere.

[0026] The present invention also relates to the use of a primer as defined herein in an oligonucleotide amplification method. It is envisaged that said oligonucleotide amplification method makes use of the vertebrate genomic nucleic acid which comprises the target sequence as a template. An oligonucleotide amplification method thereby denotes a method which is characterized by a step wherein the primer that is described herein is extended from its 3 ^" -end. "Extended from its 3 ^" -end" is explained herein elsewhere. The primer thus provides a point of initiation for polymerase-mediated template-directed synthesis of a nucleic acid complementary to the template sequence.

[0027] Oligonucleotide amplification methods that can be used in the context of the present invention are well-known to the skilled person and exemplified herein (see the items and the appended examples for a non-limiting illustration of these methods). In a preferred embodiment these methods are characterized by a method for detecting the methylation status of at least one defined 5 ^' -CpG-3 ^' (comprised by a target sequence which target sequence is comprised by a vertebrate genomic nucleic acid) comprising the steps of:

(a) extending a primer which is described herein from its 3 ^' -end using said vertebrate genomic nucleic acid as template; and

(b) (thereby) detecting the methylation status of said at least one defined 5 ^' -CpG-3 ^' .

[0028] As exemplified and documented in the appended examples, the present inventors discovered that the methylation or hydroxymethylation of the "C" of said at least one defined 5 ^' -CpG-3 ^' influences the extension reaction from the 3 ^' -end when using the primer of the present invention. The rationale is as follows, without being bound by theory. The primer of the present invention has its 3 ^" end opposite to the C of said 5 ^' -CpG-3 ^' , and comprises at said 3 ^" end a mismatched nucleotide in relation to said C of said 5 ^' -CpG-3 ^' . Said mismatched nucleotide at the utmost 3'-end of the primer does not canonically pair with the cytosine of interest, in case said cytosine is not methylated or hydroxymethylated, which negatively impairs the 3 ^' -extension reaction of said primer. The methylation/hydroxymethylation of said "C" somehow compensates for said mismatch which positively impairs the 3 ^' -extension reaction of said primer (see for example Fig. 2 for further illustration). The methylation/hydroxymethylation status of the "C" in the at least one 5 ^' -CpG-3 ^' thus influences the extension reaction from the 3 ^" -end of the primer of the invention.

[0029] Said influence can be detected by standard methods (some of them are exemplified herein), e.g. by evaluating the amount of amplified product obtained in the respective methods and/or by evaluating the velocity of reaction (e.g. the velocity of dNTP incorporation) and/or by evaluating the process capability index etc. All these methods are well-known and established.

[0030] It is also envisaged that the amplified product is detected with the vertebrate genomic nucleic acid as a template and also with at least one further template (control template) that comprises at least the target sequence comprising said at least one defined 5 ^' -CpG-3 ^' and a downstream template sequence that can be amplified (said template sequence can be identical to the template sequence of the vertebrate genomic nucleic acid sequence or not). The "C" of said at least one defined 5 ^' -CpG-3 ^' of the control template is either methylated/hydroxymethylated or not. If it is methylated/hydroxymethylated, then it may serve as a sort of "positive control" (displaying the positive effect of a methyl residue on the 3 ^' - extension when using the 3 ^' -mismatched primer of the present invention). If it is not methylated/hydroxymethylated, then it may serve as a sort of negative control (displaying the negative effect of a non-methylated/non-hydroxymethylated residue on 3 ^' -extension when using the 3 ^' -mismatched primer of the present invention).

By comparison of the 3 ^' -extension reaction when using the vertebrate genomic nucleic acid as a template with the 3 ^' -extension reaction when using the positive and/or the negative control (e.g. by comparison of the amount of the respective amplified product), it is possible to determine the methylation status of said "C" comprised in the 5 ^' -CpG-3 ^' which is comprised in the vertebrate genomic nucleic acid (see the Figures and examples for further illustration). Alternatively, it is also envisaged to compare the 3 ^' -extension reaction when using the vertebrate genomic nucleic acid as a template with a "reference value" that was established beforehand, e.g. with a positive and/or negative control. It is also envisaged to establish a calibration curve with different several positive or negative controls, which will allow displaying the methylation status on a semi-quantitative or quantitative level. For example: a set amount of identical control nucleic acid sequences is provided, all of which comprise a "C" comprised in the 5 ^' -CpG-3 ^' of interest but only a given percentage of them is methylated/hydroxymethylated at the very "C", thus resulting for example in a control template (A) comprising 5% methylated "C", a control template (B) comprising 10% methylated "C", a control template (C) comprising 20% methylated "C" etc.. These control templates display the influence of the quantitative methylation status at the very "C" on the 3 ^' -end extension reaction obtained with the primer of the invention. When comparing these values obtained with these controls (for example the calibration curve obtained therewith) with the respective value obtained with the vertebrate genomic nucleic acid as a template, then it will be possible to quantify the methylation status of said "C" comprised in the 5 ^' -CpG-3 ^' of interest in said vertebrate genomic nucleic acid.

[0031] The methods and uses and kits of the present invention may thus also be used for the quantification (or the semi-quantification) of the methylation status of a defined "C" comprised in the 5 ^' -CpG-3 ^' of interest in said vertebrate genomic nucleic acid.

[0032] Detection of the amplified products may be accomplished by using any known method. For example, the amplified nucleic acids may be associated with a surface that results in a detectable physical change, e.g., an electrical change. Amplified nucleic acids may be detected in solution phase or by concentrating them in or on a matrix (such as a gel, e.g. a polyacrylamide gel - see Fig. 3C) and detecting labels associated with them (e.g., an intercalating agent such as ethidium bromide or SYBR green or labeled primer). Other detection methods use probes complementary to a sequence in the amplified product and detect the presence of the probe:product complex, or use a complex of probes to amplify the signal detected from amplified products (e.g., U.S. Pat. No. 5,424,413). Other detection methods use a probe in which signal production is linked to the presence of the target sequence because a change in signal results only when the labeled probe binds to amplified product, such as in a molecular beacon, molecular torch, hydrolyzation probes (Taqman) or hybridization switch probe (e.g., U.S. Pat. Nos. 5,1 18,801 ). Such probes typically use a label (e.g., fluorophore) attached to one end of the probe and an interacting compound (e.g., quencher) attached to another location of the probe to inhibit signal production from the label when the probe is in one conformation ("closed") that indicates it is not hybridized to amplified product, but a detectable signal is produced when the probe is hybridized to the amplified product which changes its conformation (to "open"). Detection of a signal from directly or indirectly labeled probes that specifically associate with the amplified product indicates the presence of the target nucleic acid that was amplified.

[0033] The DNA polymerase that may be used in the context of the present invention is not particularly limited. Respective DNA polymerases are known in the art (including the respective wt Polymerases and variations thereof comprising specific mutations as described and known in the art). In preferred embodiments, the DNA polymerase is a replicative DNA polymerase, more preferably selected from the group consisting of family A DNA polymerases and family B DNA polymerases, more preferably selected from the group consisting of Taq DNA polymerase; KlenTaq DNA polymerase, Thermococcus kodakaraensis (KOD) DNA polymerase, Vent DNA polymerase, Deep Vent DNA polymerase, Tth DNA polymerase etc. to name some.

[0034] In a preferred embodiment of the uses and methods and kits of the present invention, said DNA polymerase is a protein which is at least 85%, 95%, 96%, 97%, 98%, 99% or even 100% homologous over its entire length to SEQ ID No 1 , and which comprises at a position corresponding to position 141 , 143, 628 and 705 of SEQ ID No: 1 , the amino acid A(141 ), A (143), A(628) and R(705), respectively. SEQ ID No. 1 depicts the sequence of the KOD wt polymerase. The relevant sites of mutation are indicated in bold and underlined A DNA polymerase which is 100% homologous over its entire length to SEQ ID No 1 , and which comprises at a position corresponding to position 141 , 143, 628 and 705 of SEQ ID No: 1 , the amino acid A(141 ), A (143), A(628) and R(705), respectively, was used in a method for detecting the methylation status of a given template. Said Polymerase is denoted herein KOD exo- C6 (or KOD exo- variante C6). It turned out that this polymerase scores very well in the methods of the present invention (see Figure 6). [0035] SEQ ID No.1 :

MILDTDYITEDGKPVIRIFKKENGEFKIEYDRTFEPYFYALLKDDSAIEEVKKITAERHG TWTVKR

VEKVQKKFLGRPVEVWKLYFTHPQDVPAIRDKIREHPAVIDIYEYDIPFAKRYLIDK GLVPMEGD

EELKMLAFDIETLYHEGEEFAEGPILMISYADEEGARVITWKNVDLPYVDWSTEREM IKRFLRV

VKEKDPDVLITYNGDNFDFAYLKKRCEKLGINFALGRDGSEPKIQRMGDRFAVEVKG RIHFDLY

PVIRRTINLPTYTLEAVYEAVFGQPKEKVYAEEITTAWETGENLERVARYSMEDAKV TYELGKE

FLPMEAQLSRLIGQSLWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDEKELARRRQ SYEGG

YVKEPERGLWENIVYLDFRSLYPSIIITHNVSPDTLNREGCKEYDVAPQVGHRFCKD FPGFIPSL

LGDLLEERQKIKKKMKATIDPIERKLLDYRQRAIKILANSYYGYYGYARARWYCKEC AESVTAW

GREYITMTIKEIEEKYGFKVIYSDTDGFFATIPGADAETVKKKAMEFLKYINAKLPG ALELEYEGF

YKRGFFVTKKKYAVIDEEGKITTRGLEIVRRDWSEIAKETQARVLEALLKDGDVEKA VRIVKEVT

EKLSKYEVPPEKLVIHEQITRDLKDYKATGPHVAVAKRLAARGVKIRPGTVISYIVL KGSGRIGD

RAIPFDEFDPTKHKYDAEYYIENQVLPAVERILRAFGYRKEDLRYQKTRQVGLSAWL KPKGT

[0036] The present invention thus also relates to a protein which is at least 85%, 95%, 96%, 97%, 98%, 99% or even 100% homologous over its entire length to SEQ ID No 1 , and which comprises at a position corresponding to position 141 , 143, 628 and 705 of SEQ I D No: 1 , the amino acid A(141 ), A (143), A(628) and R(705), respectively. The present invention also relates to nucleic acids encoding the protein which is at least 85% homologous over its entire length to SEQ ID No 1 , and which comprises at a position corresponding to position 141 , 143, 628 and 705 of SEQ ID No: 1 , the amino acid A(141 ), A (143), A(628) and R(705), respectively. Vectors comprising said nucleic acid are also contemplated. Host cells comprising these vectors (e.g. in order to express the respective polymerase) are also included herein.

[0037] In a further embodiment, the present invention relates to the use of a primer as defined herein and the polymerase as defined above in an oligonucleotide amplification method. In a preferred embodiment, said amplification method is for the specific amplification of a vertebrate genomic nucleic acid which comprises a target sequence which target sequence comprises at least one defined 5 ^' -CpG-3 ^' .

[0038] Another aspect of the invention is generally directed to a kit. A "kit," as used herein, typically defines a package including one or more of the primer of the invention, and/or other compositions associated with the invention, for example, a nucleic acid probe, and/or a positive control, and/or a negative control, and/or a polymerase, and/or a microarray, etc, as described herein. The kit may be directed to: determining the methylation status of at least one 5 ^' -CpG-3 ^' ; determining the methylation status of one or more selected nucleic acids molecules (for example, of genomic DNA, mitochondrial DNA, etc. as described herein); the quantification (or the semi-quantification) of the methylation status of a defined "C" comprised in the 5 ^' -CpG- 3 ^' of interest in said vertebrate genomic nucleic acid; determining the methylations status of said at least one defined 5 ^' -CpG-3 ^' which is associated with a disease (or disease state) and/or is comprised in a genetic element which is associated with a disease (or disease state); diagnostics, diagnostic analysis or for bioanalytics, or for the screening of tissue or fluids from the human or even animal body for the presence of certain methylation pattern; in vitro diagnosis of a disease of a vertebrate, which disease is associated with the methylation status of the genomic nucleic acid at said C; identifying nucleic acid molecules differentially methylated in a disease; for predicting the therapeutic response of patients suffering from a disease, which disease is associated with the methylation status of the genomic nucleic acid at said C; etc., to name some. All intensions or uses mentioned herein may be ascribed to the methods and kits of the invention.

In another embodiment of the invention, the kit further contains reagents for isolating the vertebrate genomic nucleic acid. Each of the ingredients of the kit may be provided in liquid form (e.g., in solution), or in solid form (e.g., a dried powder). In certain cases, some of the compositions may be constitutable or otherwise processable (e.g., to an active form), for example, by the addition of a suitable solvent or other species, which may or may not be provided with the kit. Examples of other compositions or components associated with the invention include, but are not limited to, solvents, surfactants, diluents, salts, buffers, emulsifiers, chelating agents, fillers, antioxidants, binding agents, bulking agents, preservatives, drying agents, antimicrobials, needles, syringes, packaging materials, tubes, bottles, flasks, beakers, dishes, frits, filters, rings, clamps, wraps, patches, containers, and the like, for example, for using, modifying, assembling, storing, packaging, preparing, mixing, diluting, and/or preserving the compositions components for a particular use. A kit of the invention may, in some cases, include instructions and/or an imprint in any form. For instance, the instructions may include instructions for the use, modification, mixing, diluting, preserving, assembly, storage, packaging, and/or preparation of the compositions and/or other compositions associated with the kit. In some cases, the instructions may also include instructions, for example, for a particular use disclosed herein. The same applies to the imprint. The instructions may be provided in any form recognizable by one of ordinary skill in the art as a suitable vehicle for containing such instructions, for example, written or published, verbal, audible (e.g., telephonic), digital, optical, visual (e.g., videotape, DVD, etc.) or electronic communications (including Internet or web-based communications), provided in any manner. The present invention thus relates to a kit comprising at least one primer as defined herein and optionally means to conduct the amplification. In a preferred embodiment, said means comprises at least the DNA polymerase as defined herein, in particular the DNA polymerase which is a protein which is at least 85%, 95%, 96%, 97%, 98%, 99% or even 100% homologous over its entire length to SEQ ID No 1 , and which comprises at a position corresponding to position 141 , 143, 628 and 705 of SEQ ID No: 1 , the amino acid A(141 ), A (143), A(628) and R(705), respectively.

[0039] In a further embodiment, the present invention relates to a kit comprising a primer as defined herein and an isolated oligonucleotide as a positive control and/or a negative control, wherein said positive control comprises the target sequence for said at least one primer including said at least one defined 5 ^' -CpG-3 ^' and wherein the C in said at least one defined 5 ^' - CpG-3 ^' is methylated or hydroxymethylated, and wherein said negative control comprises the target sequence for said at least one primer including said at least one defined 5 ^' -CpG-3 ^' and wherein the C in said at least one defined 5 ^' -CpG-3 ^' is neither methylated nor hydroxymethylated.

[0040] DNA methylation and chromatin structure are often altered in diseases, particularly in cancer. Cancer, in general, is caused by dysfunction of genes which control the cell cycle, apoptosis and migration. During carcinogenesis, oncogenes are activated and enhance division or prevent cell death. Tumor suppressor genes can be inactivated and are no longer available to stop these procedures. There are at least three pathways of gene inactivation: A mutation can disable gene function, a gene can get lost and is, thus, not available, and, lastly, a gene that is not mutated or lost is switched off by epigenetic changes. This last possibility can involve inappropriate cytosine methylation in 5 ^' -CpG-3 ^' motifs within control regions of gene expression. Over the last 40 years, various studies have shown alterations in 5- methylcytosine patterns between normal and cancer cells in human DNA. There are several major routes by which cytosine methylation can contribute to the development of cancer. The genome can be hypomethylated and this leads to genomic instability, or the promoters of tumor suppressor genes become hypermethylated which leads to silencing of these genes. Moreover, methylated CpG sites are mutation hot spots, as spontaneous deamination of 5- methylcytosine to the natural base thymine is not recognized. Lastly, methylated 5 ^' -CpG-3 ^' sites increase the rate of UV-induced mutations and the binding of some chemical carcinogens. Epigenetic silencing and genetic mutations are often recessive and require the disruption of both alleles for full expression of the changed phenotype. Three classes of hits participate in different combinations to inhibit completely the function of tumor suppressor genes. The first hit of inactivation can be a direct mutation or gene silencing by DNA methylation. The second step could be the loss of heterozygosity or DNA methylation again.

[0041] Hypermethylation is reciprocally correlated with transcription and, therefore, research has so far focused on hypermethylation of CpG islands. Moreover, this correlation is required for identification and validation of novel tumor suppressor genes. If the methylation pattern is specific for a tumor type or correlates with clinically important parameters, DNA methylation might be a useful biomarker for tumor diagnosis or risk assessment. However, the analysis of DNA methylation patterns is complicated because some changes are due to environmental influences. Additionally, ageing might be the cause of methylation accumulations at promoters. In order to possess a useful biomarker, age-associated changes in methylation have to be distinguished from alterations that predispose cancer. Clinically applicable biomarkers need to be specific and sensitive. There are many biomarkers on DNA, RNA or protein level, whereby the biomarkers based on DNA have clear advantages. DNA is more stable than RNA or protein, and methyl groups on cytosines are part of the covalent DNA which is not the case for chromatin. Furthermore, DNA methylation analysis is independent of the total amount of starting material because the ratio of methylated and unmethylated CpG sites is determined. 5- Methylcytosine represents a positive epigenetic marker that can be detected independently of expression levels and more easily than a negative signal like loss of heterozygosity. Another advantage is the theoretical reversal of epigenetic changes by treatment with pharmaceuticals, whereas genetic changes are irreversible.

[0042] In view of the above, it is envisaged that the methylations status of said at least one defined 5 ^' -CpG-3 ^' is associated with a disease (or disease state) and/or is comprised in a genetic element which is associated with a disease (or disease state), such as for example a tumor suppressor gene, a genetic element controlling the expression of a tumor associated antigen etc..

[0043] In a further embodiment, the present invention relates to the use of at least one primer of the invention for in vitro diagnosis of a disease of a vertebrate, which disease is associated with the methylation status of the genomic nucleic acid at said C. The diseases mentioned include but are not limited to neurological diseases, metabolic diseases, cardiovascular diseases, autoimmune diseases, cancer etc. The term "cancer" in an animal refers to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Often, cancer cells will be in the form of a tumor, but such cells may exist alone within an animal, or may circulate in the blood stream as independent cells, such as leukemic cells.

[0044] The methods, uses and kits of the present invention may also be used for identifying nucleic acid molecules differentially methylated in a disease. The methods/uses and kits of the present invention may for example be utilized to identify candidate tumor suppressor genes. Thus, the methods, uses and kits of the invention are particularly valuable for identifying new markers whose methylation status is linked to disease. The embodiments of the present invention may also be used for classifying epithelial and mesenchymal phenotypes in cancer (see e.g. WO2013055530 for illustration); for predicting the sensitivity of tumor cell growth to inhibition by inhibitors (see e.g. WO2013055530 for illustration).

[0045] The uses, kits and methods of the present invention may thus be used for diagnosis of diseases. Since specific alterations in the methylation status of the respective "C" comprised by at least one defined 5 ^' -CpG-3 ^' may be associated with disease state, these methods, kits and uses serve as reliable platform for diagnosis, prognosis and the analysis associated with diseases, which diseases are characterized by an altered or predictive methylation status. The term "diagnosis" is used herein to refer to the identification of a molecular or pathological state, disease or condition. The term "prognosis" is used herein to refer to the prediction of the likelihood of disease-attributable symptoms. The methods, uses and kits provided can also be used to profile populations to aid the development and application of patient-oriented treatments.

[0046] Further diseases that are contemplated in the context of the present invention are exemplified herein (see Figure 5 or the table below) but the invention is not limited thereto.

Gene/genetic Element Cancer/ diseases

APC Prostate, colon, lung, bladder

A Prostate

BMAL1 Leukaemia, lymphoma

BRCA1 Breast, ovarian

CDH1 Breast, prostate

CDH11 Colon, breast, oesophagus, gastric, liver

CDH13 Lung, head and neck

CDKN2A Lymphoma, colon, stomach, prostate

CDKN2B Leukaemia DAPK1 Lung, head and neck, bladder

EMP3 Glioma

ES 1 Breast

GSTP1 Prostate, liver, lung

IGFBP3 Colon, lung, ovarian, prostate

LGALS3 Prostate

MASPIN Pancreas

MGMT Colon, glioma, lymphoma, prostate, lung miR-148a Metastasis

miR-34b and miR-34c Metastasis

miR-9 Metastasis

miR-200s Colon, bladder, squamous cell carcinoma

MLH1 Colon, endometrium, stomach

N0RE1A Colon, liver, lung, thyroid

NSD1 Glioma, neuroblastoma

PYCARD Glioma, breast, colon, gastric, lung

RARB Breast, colon, prostate

RASSF1A Breast, ovarian, lung, prostate, colon

RBP1 Lymphoma, gastric, squamous cell carcinoma

RIZ1 Leukaemia, liver, thyroid, gastric, prostate

S100P Pancreatic

SHOX2 Lung

SEPT9 Colon

SFRP1 Lung, liver, kidney, leukemia

SFRP2 Colon

SNCG Breast, ovarian

SOCS1 Liver

TFPI2 Colon

THBS1 Glioma

TIG1 Prostate

TIMP2 Prostate

TP73 Lymphoma

TSHR Thyroid

VHL Kidney

WIF1 Colon

WRN Colon

AC051635.7 Bladder

PRDM14 Bladder

DMRT2 Bladder

CYP1 Bl Bladder SOX1 Bladder

AGT 1 Colorectal

WNT2 Colorectal

SLIT2 Colorectal

VIM Colorectal

RASSF1A Breast

HIN-1 Breast

RAR-beta Breast

cyclin D2 Breast

Twist Breast

NID2 oral cavity squamous cell carcinoma

HOXA9 oral cavity squamous cell carcinoma

FOXE1 Breast Cancer (diagnosis and/or prognosis)

CLDN5 Breast Cancer (diagnosis and/or prognosis)

RUNX3 Breast Cancer (diagnosis and/or prognosis)

DUSP4/MKP2 glioma and other tumors

BCL2 human colorectal cancer

BDNF human colorectal cancer

CACNA1G human colorectal cancer

CALCA human colorectal cancer

CRABP1 human colorectal cancer

DLEC1 human colorectal cancer

GATA3 human colorectal cancer

HOXA1 human colorectal cancer

IGF2 human colorectal cancer

KL human colorectal cancer

NEUROG1 human colorectal cancer

NR3C1 human colorectal cancer

RUNX3 human colorectal cancer

SOCS1 human colorectal cancer

FABP5 Melanoma

IFFOl-M Ovarion Cancer

PCDHGA12 Lung cancer

EPB41L3 cervix cancer

HOXA9 bladder cancer

ZNF154 bladder cancer

POU4F2 bladder cancer

FGFR1 drug response prediction Tamoxifen, Breast cancer

PSA drug response prediction Tamoxifen, Breast cancer

CGA drug response prediction Tamoxifen, Breast cancer

PTGS2 drug response prediction Tamoxifen, Breast cancer

MSMB drug response prediction Tamoxifen, Breast cancer

TP53 drug response prediction Tamoxifen, Breast cancer

CAD Ml HPV-induced cervical premalignant lesion MAL HPV-induced cervical premalignant lesion

ACSS3 Cervix Carcinom

ADCYAP1 Cervix Carcinom

HOXA11 Cervix Carcinom

MINT 17 Melanoma and Breast Cancer

MINT31 Melanoma and Breast Cancer

WIFI Melanoma and Breast Cancer

TFPI2 Melanoma and Breast Cancer

ASSFIA Melanoma and Breast Cancer

SOCS1 Melanoma and Breast Cancer

NEP Alzheimer's disease, Hypermethylation

15qll.2-ql3 Angelman syndrome (Imprinting defect)

FXN Friedrich's ataxia, Hypermethylation

PADI2 Multiple sclerosis, Hypomethylation

15qll.2-ql3 Prader-Willi syndrome, Imprinting defect

DR3 and LI Rheumatoid arthritis, Aberrant methylation

[0047] Other possible indications are exemplified below (although the present invention is in no way limited thereto):

target indication

CD3 Identification of IL17 positive T-cells

FOXP3 Identification of IL17 positive T-cells

GAPDH Identification of IL17 positive T-cells

EDARADD estimation of age

TOMILI estimation of age

NPTX2 estimation of age

FMR1 prediction of treatment of Fragile X Syndrome (FXS)

ZNF447 Determination of the presence of cancer cells in a biological sample

COL4A2 Determination of the presence of cancer cells in a biological sample

AOX1 Determination of the presence of cancer cells in a biological sample

DUSP26 Determination of the presence of cancer cells in a biological sample

EDIL3 Determination of the presence of cancer cells in a biological sample

EFHD1 Determination of the presence of cancer cells in a biological sample

Determination of the presence of cancer cells in a biological

ELMOl sample Determination of the presence of cancer cells in a biological

STOX2 sample

Determination of the presence of cancer cells in a biological

Zarl sample

fetal methylation marker (for discrimination of maternal

ASSF1A and fetal DNA)

fetal methylation marker (for discrimination of maternal

APC and fetal DNA)

fetal methylation marker (for discrimination of maternal

CASP8 and fetal DNA)

fetal methylation marker (for discrimination of maternal

RARB and fetal DNA)

fetal methylation marker (for discrimination of maternal

SCGB3A1 and fetal DNA)

fetal methylation marker (for discrimination of maternal

DAB2IP and fetal DNA)

fetal methylation marker (for discrimination of maternal

PTPN6 and fetal DNA)

fetal methylation marker (for discrimination of maternal

THY1 and fetal DNA)

fetal methylation marker (for discrimination of maternal

TMEFF2 and fetal DNA)

fetal methylation marker (for discrimination of maternal

PYCARD and fetal DNA)

[0048] It is well known that the methylation status may also be used to evaluate the age of a subject (WO 2012162139) or to discriminate fetal from maternal DNA (US 201 10143342). Further applications of the methods and uses and kits of the present invention are disclosed in WO2001068912. It will be understood however, that the uses, kit and methods of the present invention may be used in all thinkable situations where it is already known or assumed that the methylation status of a defined "C" is of interest, for example because it is indicative of a disease.

[0049] In a preferred embodiment of the present invention (including the uses, kits and methods of the invention) the present invention aims at determining or diagnosing the methylation status of at least one of the genes selected from BRCA1 , MGMT PYCARD, RASSF1A, SHOX2 and/or SEPT9.

[0050] The present invention further relates to the use of at least one primer as defined herein, for the stratification of patients suffering from a disease, which disease is associated with the methylation status of the genomic nucleic acid at said C. It is envisaged that the above embodiment makes use of the vertebrate genomic nucleic acid which comprises the target sequence as a template together with said primer. [0051] The present invention also relates to the use of at least one primer as defined herein, for predicting the therapeutic response of patients suffering from a disease, which disease is associated with the methylation status of the genomic nucleic acid at said C. It is envisaged that the above embodiment makes use of the vertebrate genomic nucleic acid which comprises the target sequence as a template together with said primer.

[0052] In a further embodiment, the present invention relates to a primer as defined herein for use in a method of the invention and in particular for determining the methylation status of a vertebrate genomic nucleic acid at said C and/or for (in vitro) diagnosis of a disease of a vertebrate, which disease is associated with the methylation status of the genomic nucleic acid at said C. It is envisaged that the above embodiment makes use of the vertebrate genomic nucleic acid which comprises the target sequence as a template together with said primer.

[0053] The present invention also relates to the following items and the corresponding embodiments.

Item 1 A method for directly detecting methylation or hydroxymethylation of a cytosine residue of interest in a DNA molecule, comprising the steps of:

(a) providing a primer having its 3'-end opposite of the cytosine residue of interest and having at said 3'-end a mismatched base;

(b) performing a specific DNA polymerase reaction with said primer using said DNA molecule as template; and

(c) detecting said methylation or hydroxymethylation, wherein methylation or hydroxymethylation is indicated by an increased efficiency of said specific DNA polymerase reaction compared to a corresponding DNA polymerase reaction performed with said primer using a corresponding DNA molecule, wherein the cytosine residue of interest is not methylated or hydroxymethylated, as template.

Item 2. The method of item 1 , wherein the mismatched base is selected from the group consisting of adenine, cytosine, thymine, uracil and modifications thereof.

Item 3. The method of item 1 or claim 2, wherein said specific DNA polymerase reaction is selected from the group consisting of primer extension, rolling circle extension (RCA) and polymerase chain reaction (PCR).

Item 4. The method of item 3, wherein said PCR is quantitative real-time PCR (qRT-PCR). Item 5. The method of any one of items 1 to 4, wherein the DNA polymerase used for said specific DNA polymerase reaction is selected from the group consisting of family A DNA polymerases and family B DNA polymerases.

Item 6. The method of item 5, wherein the DNA polymerase is selected from the group consisting of KlenTaq DNA polymerase, KOD DNA polymerase, Vent DNA polymerase, and Deep Vent DNA polymerase.

Item 7. The method of any one of items 1 to 6, wherein the increased efficiency of the specific DNA polymerase reaction indicating methylation or hydroxymethylation of the DNA molecule is an increased efficiency by 1 to 30 cycles, preferably 5 to 20 cycles, more preferably 10 to 20 cycles.

The present invention thus also relates to a method for directly detecting methylation or hydroxymethylation of a cytosine residue of interest in a DNA molecule, comprising the steps of:

(a) providing a primer having its 3'-end opposite of the cytosine residue of interest and having at said 3'-end a mismatched base;

(b) performing a specific DNA polymerase reaction with said primer using said DNA molecule as template; and

(c) detecting said methylation or hydroxymethylation, wherein methylation or hydroxymethylation is indicated by an increased efficiency of said specific DNA polymerase reaction compared to a corresponding DNA polymerase reaction performed with said primer using a corresponding DNA molecule, wherein the cytosine residue of interest is not methylated or hydroxymethylated, as template.

As used in the context of the items, the terms "directly detecting" or "direct detection" relate to the fact that with the method of the present invention as described in the items, methylation or hydroxymethylation of a cytosine residue of interest in a DNA molecule can be directed without the need for any pretreatment or chemical modification of the DNA molecule. Accordingly, the method of the present invention as described in the items is significantly less time-, labor- and cost-intensive compared to methods known in the art. Moreover, the method of the present invention as described in the items is much less prone to errors and allows the analysis of very small amounts of sample material.

The primer provided in step (a) of the method of the present invention as described in the items is specifically designed for the analysis of a particular cytosine residue of interest in a known DNA molecule. In particular, said primer binds to the DNA molecule in a manner that its 3'-end is opposite of the cytosine residue and said 3'-end has a mismatched base in respect to the cytosine of interest. Said mismatched base at the 3'-end of the primer does not canonically pair with the cytosine of interest, in case said cytosine is not methylated or hydroxymethylated, thus impairing the specific DNA polymerase reaction with said primer using said DNA molecule as template. However, said mismatched base at the 3'-end of the primer is thought to pair in a non-canonical manner with the cytosine of interest, in case said cytosine is methylated or hydroxymethylated, thus allowing a more efficient specific DNA polymerase reaction with said primer using said DNA molecule as template (Fig. 2). Preferably the mismatched based is selected from the group consisting of adenine, cytosine and thymine, and modifications thereof. The design and generation of suitable primers is known in the art. As already described herein elsewhere, primers can be labeled with a detectable marker as known in the art, e.g. with a radioactive or dye label. The method of the present invention as described in the items is a general method and can advantageously be used in every conceivable sequence context. In particular examples of the present invention, the primer is selected from the group of primers as shown in SEQ ID NOs. 2 to 5 (SEQ ID NO. 2: TTG CTC CCG TCG GCG CTT CTT TCA; SEQ ID NO. 3: GTT TCT CCA GTT TCT TTT CTC A; SEQ ID NO. 4: GTT TCT CCA GTT TCT TTT CTC C; SEQ ID NO. 5: GTT TCT CCA GTT TCT TTT CTC T).

The term "modifications thereof" as used herein in the context of "adenine, cytosine and thymine, uracil, and modifications thereof" as described in the items, relates to any adenine, cytosine and thymine derivatives that retain the characteristics of mismatching with methylated or hydroxymethylated cytosine.

In a particular embodiment of the method of the present invention as described in the items, the mismatched base is an artificial nucleobase that has the characteristic of mismatching with methylated or hydroxymethylated cytosine. Respective artificial nucleobases are not particularly limited and are known in the art.

The specific DNA polymerase reaction performed in step (b) and/or (c) of the method of the present invention as described in the items is not particularly limited, provided that it allows the discrimination between unmodified and methylated or hydroxymethylated cytosine residues. Suitable DNA polymerase reactions include established standard methods and are known in the art. In preferred embodiments, the specific DNA polymerase reaction is selected from the group consisting of primer extension, rolling circle amplification (RCA) and PCR-based methods such as quantitative real-time PCR (qRT-PCR). In case the specific DNA polymerase reaction is a primer extension reaction, said primer extension reaction is preferably performed for 10 to 90 seconds. In case the specific DNA polymerase reaction is a PCR-based method, a suitable additional primer (i.e. reverse primer) is used as known in the art. The DNA polymerase used for the specific DNA polymerase reaction is not particularly limited. Respective DNA polymerases are known in the art. In preferred embodiments, the DNA polymerase is a replicative DNA polymerase, more preferably selected from the group consisting of family A DNA polymerases and family B DNA polymerases, more preferably selected from the group consisting of KlenTaq DNA polymerase, Thermococcus kodakaraensis (KOD) DNA polymerase, Vent DNA polymerase, and Deep Vent DNA polymerase.

In step (c) of the method of the present invention as described in the items, methylation or hydroxymethylation of the cytosine residue of interest is indicated by an increased efficiency of said specific DNA polymerase reaction compared to a corresponding DNA polymerase reaction performed with said primer using a corresponding DNA molecule, wherein the cytosine residue of interest is not methylated or hydroxymethylated, as template. In other words, the efficiency of the specific DNA polymerase reaction is assessed in comparison to a corresponding unmodified DNA molecule, i.e. a DNA molecule wherein the cytosine residue of interest is neither methylated nor hydroxymethylated. The term "corresponding DNA molecule" as used in this context relates to a DNA molecule having the same sequence as the DNA molecule to be analyzed at least in the region of primer binding and the upstream region that is replicated in the DNA polymerase reaction. Preferably, the increased efficiency of the specific DNA polymerase reaction indicating methylation or hydroxymethylation of the DNA molecule is an increased efficiency by 1 to 30 cycles, preferably 5 to 20 cycles, more preferably 5 to 15 cycles. Also preferably, the increased efficiency of the specific DNA polymerase reaction indicating methylation or hydroxymethylation of the DNA molecule is an increased efficiency by 1 to 30, 5 to 30, 5 to 25, 10 to 25, 10 to 20, or 15 to 20 cycles. Alternatively, methylation or hydroxymethylation of the cytosine residue of interest is indicated by an increased efficiency of primer extension reactions which can be quantified in an absolute manner.

[0054] Methods of the present invention as described in the items provide a means for the direct detection of methylated or hydroxymethylated cytosine residues of interest in a DNA molecule in a fast, simple, and accurate manner. Said methods can be conducted without a bisulphite pretreatment of the DNA templates, i.e. these methods do not need a pretreatment, such as a bisulphite pretreatment or chemical modification of the DNA molecule to be analyzed. Therefore, it is less prone to errors as compared to methods known in the art, and allows the analysis of smallest amounts of sample material. Moreover, said method can be performed using well established standard methods for the DNA polymerase reaction. [0055] It must be noted that as used herein, the singular forms "a", "an", and "the", include plural references unless the context clearly indicates otherwise. Thus, for example, reference to "a reagent" includes one or more of such different reagents and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.

[0056] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0057] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term "comprising" can be substituted with the term "containing" or sometimes when used herein with the term "having".

[0058] When used herein "consisting of" excludes any element, step, or ingredient not specified in the claim element. When used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0059] As described herein, "preferred embodiment" means "preferred embodiment of the present invention". Likewise, as described herein, "various embodiments" and "another embodiment" means "various embodiments of the present invention" and "another embodiment of the present invention", respectively.

[0060] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. The figures show:

Figure 1 : Modifications of cytosine

A) Unmodified cytosine. B) 5-Methylcytosine. C) 5-Hydroxymethylcytosine. Figure 2: Basic principle of the method of the present invention

A) Schematic depiction of primer template complex bearing a mismatched base pair at the 3'- end of the primer opposite of the cytosine of interest in the DNA template. If the cytosine in the template is methylated or hydroxymethylated, DNA polymerases can extend the primer significantly more efficient than in the case with the unmethylated cytosine. B) Exemplary partial DNA sequence of primer, which comprises at said 3 ^" end a mismatched nucleotide (X) in relation to said C of said 5 ^' -CpG-3 in the template.

Figure 3: Direct detection of methylcytosine by primer extension

A) DNA sequences used for primer extension reactions. The C ^* indicates the cytosine of interest and is either methylated or unmethylated. The primer ends with an adenine to generate a mismatch opposite of the cytosine of interest in the template. B) Structures of cytosine and 5-methylcytosine. C) Primer extension reactions of the mismatched primer opposite a methylated or unmethylated cytosine in the template. A 24 nt radioactive labeled primer was used. Full-length product is at 31 nt. The 32 nt product is formed by a non- templated nucleotide addition to the 3'-termini of the blunt-ended DNA strands and has been observed before for KlenTaq DNA polymerase. Reaction products are separated by denaturing PAGE. Reactions for methylated and unmethylated template were started in parallel and stopped after certain time periods. Clearly more product is formed with the methylated template compared to the unmethylated. D) Quantification of extended primer. The ratio of extended to unextended primer was determined with Quantity One software.

Figure 4: Direct detection of methylcytosine by quantitative real-time PCR

A) DNA sequences of the used forward primer (Pfor), reverse primer (Prev) and template (temp.). The C ^* in the template indicates the cytosine of interest and is either methylated, hydroxymethylated or unmethylated. The N in the forward primer stands for adenine, cytosine, guanine, thymine, or modifications thereof. In the case of adenine, cytosine, thymine, or modifications thereof the primer is mismatched at the 3'-end. For guanine the complete primer is matched. B) Real-time PCR curves for all possible primer template combinations. Curves for reactions in presence of a methylcytosine are shown in a dashed line, curves for the unmethylated template in a black line. The used primer is named in the headline of each graph. No discrimination is detected for the matched primer G (upper left corner). For the three mismatch primers clear discrimination between methylated and unmethylated cytosine in the template is visible.

Figure 5: non-exclusive list of hypermethylated genes in cancer

Derived from Nature Reviews/Genetics, Vol 13, October 2012; pp. 679

Figure 6 exemplary comparisons of KOD-exo- wt and KOD exo- C6 when used in the context of the present invention

Blue curve: unmethylated template (arrow); red curve: methylated template

The present invention will now be further illustrated in the following examples without being limited thereto.

Examples

Reagents and Instruments:

Oligonucleotides were purchased from Thermo Fisher Scientific or Metabion, Germany. dNTPs were either from Roche (primer extensions) or Fermentas (quantitative real-time PCR). The KlenTaq DNA polymerase was overexpressed in E. coli and purified with Ni-IDA as known in the art. Enzyme purity and quantity were determined by SDS-PAGE using an albumin standard dilution curve. Quantitative real-time PCR was performed on a Chromo4 instrument from Bio- Rad. SYBRgreen I was purchased from Fluka. Denaturing PAGE was analyzed with a Molecular Imager Fx from Bio-Rad.

Example 1 :

Primer extension assay with methylated and unmethylated DNA template

Reaction mixtures (20 μΙ_) contained 50 mM Tris-HCI (pH 9.2), 16 mM (NH ₄ ) ₂ S0 ₄ , 0.1 % Tween20, 2.5 mM MgCI ₂ , 400 nM KlenTaq DNA polymerase, 150 nM primer (24 nt, 5'- [ ³² P]d(TTG CTC CCG TCG GCG CTT CTT TCA)-3'], SEQ ID NO: 2, and 200 nM template (34 nt, 5'-d(GGC AAC GAG GGC AGC CGC GAA GAA AG ^Me C ATC CGG C)-3') (Fig. 3 A).

After an initial denaturation and annealing step (95°C for 2 min, 0.5°C/s cooling to 40°C), a temperature of 72°C was applied and the reaction was started by addition of 400 nM dNTPs. After different times (10 to 90 s) of incubation, the reactions were quenched by addition of stop solution (80% formamide, 20 mM EDTA). Product mixtures were separated by 12% denaturing PAGE and analyzed by phosphorimaging.

Results can be taken from Fig. 3 C, showing that clearly more product is formed with the methylated template compared to the unmethylated template.

Example 2:

Quantitative real-time PCR with methylated and unmethylated DNA template Reaction mixtures (20 μΙ_) contained 50 mM Tris-HCI (pH 9.2), 16 mM (NH ₄ ) ₂ S04, 0.1 % Tween20, 2.5 mM MgCI ₂ , 250 μΜ of each dNTP, 0.6x SYBRgreen I and 200 nM KlenTaq DNA polymerase. As templates, either RT-Epi90C [60 pM, 90 nt, 5'-d(GGG GCA GAG CGA GCT CCC GAG TGG GTC TGG AGC CGC GGA GCT GGG CGG GGG CGG GAA GGA GGT AGC GAG AAA AGA AAC TGG AGA AAC TCG)-3'] or RT Epi90MeC [60 pM, 90 nt, 5'-d(GGG GCA GAG CGA GCT CCC GAG TGG GTC TGG AGC CGC GGA GCT GGG CGG GGG CGG GAA GGA GGT AG ^Me C GAG AAA AGA AAC TGG AGA AAC TCG) were used (Fig. 4 A). Both templates had the same sequence except of the methylation pattern at the indicated position.

Primers were used in a concentration of 750 nM each. The reverse Primer RT-Epi22Prev [5'- d(GCA GAG CGA GCT CCC GAG TG)-3'] was used together with one of the following forward Primers RT-Epi22Afor [5'-d(GTT TCT CCA GTT TCT TTT CTC A)-3'; SEQ ID NO: 3], RT- Epi22Cfor [5'-d(GTT TCT CCA GTT TCT TTT CTC C)-3'; SEQ ID NO: 4], RT-Epi22Gfor [5'- d(GTT TCT CCA GTT TCT TTT CTC G)-3'] or RT-Epi22Tfor [5'-d(GTT TCT CCA GTT TCT TTT CTC T)-3'; SEQ ID NO: 5] (Fig. 4 A). All forward primers differed only at the last nucleotide at the 3' end.

After an initial denaturation step (95°C for 3 min), the product was amplified by 50 PCR cycles (95°C for 15 s, 55°C for 10 s and 72°C for 15 s), and analyzed by melting curve measurement from 55° to 95°C with a read every 0.5°C.

Results can be taken from Fig. 4 B, showing that no discrimination between methylated and unmethylated template is seen for the matched primer (having a G at the 3'-end), whereas a clear discrimination can be seen for the mismatched primers.