WO2014019595A1

WO2014019595A1 - Method for the detection of dna methylation and hydroxymethylation

Info

Publication number: WO2014019595A1
Application number: PCT/EP2012/003279
Authority: WO
Inventors: Andreas Marx; Matthias DRUM; Katharina STREICHERT
Original assignee: Universität Konstanz
Priority date: 2012-08-01
Filing date: 2012-08-01
Publication date: 2014-02-06
Also published as: WO2014020124A1

Abstract

The present invention 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.

Description

METHOD FOR THE DETECTION OF DNA METHYLATION AND HYDROXYMETHYLATION

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. 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 mammals, e.g. they are responsible for X-inactivation, imprinting and the development of primordial germ cells. Furthermore, DNA methylation plays a crucial role in cancer. Further, a recent study showed that the methylation status of the glucocorticoid receptor of adolescent children is influenced by their mother's experience of intimate partner violence during pregnancy.

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 solely found as symmetrical 5-methylations of the dinucleotide CpG within 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 can not 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. The definition of a CpG island is a CG content of more than 50%, an observed-expected ratio of more than 0.6 and a minimum size of 200 base pairs. 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.

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.

DNA methylation of CpG islands within 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 CpG methylation directly disturbs the binding of transcriptional regulators to their appropriate DNA sequences. Another possibility could be the recruitment of methyl-CpG 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 CpG sites might correlate better with gene expression than the methylation state of the whole CpG island. 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 CpG 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 CpG 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.

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. Moreover the specimen should be obtained through minimally invasive procedures. 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.

5-Hydroxymethylcytosine (Fig. 1 C) was first discovered in the bacteriophages 12, 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 leukaemia, was identified as a 2- oxoglutarate- and Fe(l Independent enzyme that catalyses 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 above. 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.

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 NaHS0₃ 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 CpG 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 CpG 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-methyIenesulfonate 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.

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.

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.

Accordingly, the technical problem underlying the present invention is to provide a fast, easy and accurate method for the direct detection of cytosine methylation or hydroxymethylation in a DNA molecule.

The solution to the above technical problem is achieved by the embodiments characterized in the claims.

In particular, the present invention 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 herein, the terms "directly detecting" or "direct detection" relate to the fact that with the method of the present invention, 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 is significantly less time-, labor- and cost-intensive compared to methods known in the art. Moreover, the method of the present invention 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 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. 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 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. 1 to 4. (SEQ ID NO. 1 : TTG CTC CCG TCG GCG CTT CTT TCA; SEQ ID NO. 2: GTT TCT CCA GTT TCT TTT CTC A; SEQ ID NO. 3: GTT TCT CCA GTT TCT TTT CTC C; SEQ ID NO. 4: GTT TCT CCA GTT TCT TTT CTC T).

The term "modifications thereof as used herein in the context of "adenine, cytosine and thymine, and modifications thereof, 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, 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 (c) of the method of the present invention 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, 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.

The method of the present invention provides 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 method does not need any 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.

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) Partial DNA sequence of primer and template depicting the crucial mismatch. The X in the primer can either be adenine, cytosine or thymine, or modifications thereof. All three possible primers generate a mismatch at the 3' end opposite of the cytosine of interest 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.

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 (NH₄)₂S0 , 0.1 % Tween20, 2.5 mM MgCI₂, 400 nM KlenTaq DNA polymerase, 150 nM primer (24 nt, 5'-[³²P]d(TTG CTC CCG TCG GCG CTT CTT TCA)-3 ], SEQ ID NO: 1 ), and 200 nM template (34 nt, 5'-d(GGC AAC GAG GGC AGC CGC GAA GAA AG^MeC 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 (NH₄)₂S0 , 0.1% Tween20, 2.5 mM MgCI₂, 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 ^eC 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: 2], RT-Epi22Cfor [5'-d(GTT TCT CCA GTT TCT TTT CTC C)-3'; SEQ ID NO: 3], 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: 4] (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.

Claims

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

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

3. The method of claim 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).

4. The method of claim 3, wherein said PCR is quantitative real-time PCR (qRT-PCR).

5. The method of any one of claims 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.

6. The method of claim 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.

7. The method of any one of claims 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.