Detection of methylation status

Abstract

The present invention relates to a method for determining the epigenetic modification status of at least one nucleotide of interest (NOI) in a target nucleic acid sequence of interest. The present invention is based on the discovery that certain oligonucleotides containing hydrophobic nucleotides have significantly different melting temperatures for epigenetically modified target nucleic acids compared to unmodified target nucleic acids. The method has multiple applications, for example, in diagnostics. The present invention also provides oligonucleotides, particularly base primers, for use in such methods.

Description

[Technical Field]

[0001] Background technology Methylation is an example of epigenetic modification of DNA in which a methyl group is added to one of the four DNA bases. Most commonly, the methyl group is added to cytosine at position 5 of the nucleic acid base. DNA methylation in promoter regions is involved in the regulation of gene expression.

[0002] As cancer progresses, several genes are silenced by hypermethylation of their promoters, thereby suppressing gene transcription. While methylation of specific genes can be used as a prognostic factor for cancer, it can also be used to select the optimal treatment for a patient. In glioblastoma, a type of brain tumor, methylation of the MGMT (O6-methylguanine-DNA methyltransferase) promoter is correlated with a better prognosis and a more efficient response to alkylating chemotherapy such as temozolomide (TMZ). Therefore, glioblastoma patients are often tested for MGMT methylation.

[0003] Several methods are available for detecting methylation, with the gold standard method requiring bisulfite pretreatment of DNA. This pretreatment converts unmethylated cytosine to uracil, while methylated cytosine remains unchanged. Bisulfite treatment is time-consuming, requires large amounts of DNA, and tends to produce false results because too short a reaction time can result in incomplete conversion of unmethylated cytosine, while too long a reaction time can result in conversion of methylated cytosine. This leads to a risk of both false positive and false negative results. Methods that do not use bisulfite have also been developed, but they all require a different conversion or are not PCR-based. Bisulfite conversion kits are commercially available, but typically require at least 15 operating steps.

[0004] Hydrophobic nucleotides, such as intercalated nucleic acids (INA®), contain a hydrophobic moiety, such as an intercalator. An intercalator is a flat, conjugated or heteroaromatic ring system that can participate in the stacking of DNA or RNA double helixes. Hydrophobic nucleotides do not participate in Watson-Crick base pairing. When an oligonucleotide incorporating a hydrophobic nucleotide binds to an unmodified DNA sequence, the hydrophobic moiety itself is positioned at the center of the DNA helix. Interactions between hydrophobic nucleotides and DNA bases increase the stability of double-stranded DNA. [Overview of the project]

[0005] Simple methods for detecting epigenetic modifications of nucleic acid bases, such as analyzing methylation of nucleic acid bases without converting the nucleic acid, are beneficial because they allow for faster detection of methylated nucleic acids, eliminate sample loss during conversion steps, and avoid uncertainty based on conversion that may be incomplete or nonspecific. In clinical practice, early detection of epigenetic modifications such as methylation status can lead to faster patient diagnosis, potentially enabling earlier initiation of treatment. Better methods may allow for more accurate quantification of methylation levels and expand the use of methylation status, for example, in the treatment and monitoring or diagnosis of cancer.

[0006] The present invention provides a very rapid method for detecting epigenetic modifications in nucleic acids, such as methylated DNA. These methods are based on the discovery that synthetic oligonucleotides containing at least one hydrophobic nucleotide can have a remarkably high affinity (melting temperature) difference (as designed) between DNA with epigenetic modifications and DNA without epigenetic modifications. This is particularly true when the hydrophobic nucleotide is positioned within the synthetic oligonucleotide and intercalates between a potentially epigenetically modified nucleotide (such as methylation) and the nucleotide immediately 3' to it on the target nucleic acid.

[0007] The method of the present invention can be used to detect epigenetic modifications of any nucleotide in any type of nucleic acid, but this method is particularly useful for detecting cytosine methylation in DNA. In such cases, the synthetic oligonucleotide is preferably designed such that the hydrophobic nucleotide intercalates between the potentially methylated cytosine and the nucleotide immediately 3' thereto (most often guanine).

[0008] This surprisingly high melting temperature difference can be utilized to easily detect the methylation state of nucleic acids using various methods. For example, the methylation state of nucleic acids can be detected by a simple PCR method. Importantly, in the method of the present invention, no pretreatment of the nucleic acid is required. Thus, nucleic acid methylation can be detected by one-step PCR by designing appropriate primers containing hydrophobic nucleotide(s).

[0009] In a preferred embodiment of the present invention, the method is carried out using a type of primer called a base primer, which is described in more detail below.

[0010] This is one aspect of the present invention that provides a method for determining the methylation state of at least one nucleotide of interest (NOI) in a target nucleic acid sequence of interest, the target nucleic acid sequence comprising a target anchor sequence containing the NOI, and the method comprising a. providing an oligonucleotide comprising an anchor sequence (An), the anchor sequence being at least 50% complementary to the target anchor sequence, the anchor sequence comprising at least one hydrophobic nucleotide (H) located between a nucleotide complementary to the NOI and a nucleotide in the oligonucleotide immediately 5' thereto; b. incubating the oligonucleotide with the target nucleic acid of interest at a temperature higher than the melting temperature between the oligonucleotide and the target nucleic acid of interest when the NOI is not methylated; c. A step of detecting whether the oligonucleotide anneals to the target nucleic acid of the desired type, This includes the step of determining the methylation state, During the ceremony i. Hydrophobic nucleotides (H) have a structure XYQ It has, in the formula, X is a skeletal monomer unit that can be incorporated into the backbone of a nucleotide or nucleotide analog, or a nucleic acid or nucleic acid analog. Q is an intercalator that does not participate in Watson-Crick hydrogen bonding. Y is a linker portion that connects a nucleotide, nucleotide analog, or skeletal monomer unit to an intercalator.

[0011] Furthermore, this is one aspect of the present invention that provides a method for determining the methylation state of at least one target nucleotide (NOI) in a target unmodified target nucleic acid sequence, wherein the target nucleic acid sequence includes a target anchor sequence containing the NOI, and the method is a. A step of providing an oligonucleotide comprising an anchor sequence (An), wherein the anchor sequence is at least 50% complementary to a target anchor sequence, and the anchor sequence comprises at least one hydrophobic nucleotide (H) located between a nucleotide complementary to the NOI and the nucleotide immediately to its 5' side; b. If the NOI is not methylated, the step is to incubate the oligonucleotide with the target nucleic acid at a temperature higher than the melting temperature between the oligonucleotide and the target nucleic acid sequence of interest. c. A step of detecting whether the oligonucleotide anneals to the target nucleic acid of the desired value, This includes the step of determining the methylation state, During the ceremony, i. Hydrophobic nucleotides (H) have a structure XYQ It has, in the formula, X is a skeletal monomer unit that can be incorporated into the backbone of a nucleotide or nucleotide analog, or a nucleic acid or nucleic acid analog. Q is an intercalator that does not participate in Watson-Crick hydrogen bonding. Y is a linker portion that connects a nucleotide or nucleotide analog or backbone monomer unit to an intercalator. ii. Oligonucleotides have the structure 5'-An-Lp-St-3', in which, An is an anchor array; Lp is a loop sequence that is not complementary to the target nucleic acid sequence of interest, and the loop sequence consists of a single nucleic acid sequence that can form a protruding structure such as a loop structure or a stem structure, or consists of two or more nucleic acid sequences that can at least partially hybridize with each other to form a complex that can form a protruding structure such as a loop structure or a stem structure; St is a starter sequence that can hybridize to a target starter sequence, where the target starter sequence is a target nucleic acid sequence located 5' relative to the target anchor sequence.

[0012] In another aspect, the present invention provides a method for determining whether an individual is at risk of having a clinical condition, or the risk of an individual becoming at risk of developing a clinical condition, wherein the clinical condition is related to the methylation status of NOI in the nucleic acid of interest, and the method is a. To provide samples obtained from individuals; b. Determining the methylation status of NOI in a sample by performing the methods disclosed herein, wherein the methylation status indicates the presence or risk of developing a clinical condition.

[0013] In some embodiments, the present invention provides a method for determining the potential therapeutic effect of a clinical condition in an individual requiring it, wherein the therapeutic effect of the clinical condition is related to the methylation state of NOI in the nucleic acid of interest, and the method provides a. To provide samples obtained from individuals; b. Determining the methylation status of NOI in a sample by performing the methods disclosed herein, wherein the methylation status indicates the effect of different treatment regimens on a clinical condition.

[0014] In some embodiments, oligonucleotides comprising or consisting of the following general structure are provided: 5'(N) n -HG-(N) m -HG-(N) p -3' (wherein N is any nucleotide or nucleotide analogue; G is the nucleotide guanine; n is a non-negative integer; m is an integer greater than or equal to 1; p is a non-negative integer; H is a hydrophobic nucleotide, Here, the hydrophobic nucleotide has the following structure; XYQ During the ceremony, X is a skeletal monomer unit that can be incorporated into the backbone of a nucleotide or nucleotide analog, or a nucleic acid or nucleic acid analog. Q is an intercalator that does not participate in Watson-Crick hydrogen bonding; Y is a linker portion that connects a nucleotide or nucleotide analog or backbone monomer unit to an intercalator.

[0015] In yet another embodiment, oligonucleotides comprising or consisting of the following general structure are provided: 5'(N) n -CGH-(N) m -CGH-(N) p -3' (wherein N is any nucleotide or nucleotide analogue; C is the nucleotide cytosine; G is the nucleotide guanine; n is a non-negative integer; m is an integer greater than or equal to 1; p is a non-negative integer; H is a hydrophobic nucleotide, Here, the hydrophobic nucleotide has the following structure; XYQ During the ceremony, X is a skeletal monomer unit that can be incorporated into the backbone of a nucleotide or nucleotide analog, or a nucleic acid or nucleic acid analog. Q is an intercalator that does not participate in Watson-Crick hydrogen bonding; Y is a linker portion that connects a nucleotide or nucleotide analog or backbone monomer unit to an intercalator.

[0016] The present invention is further defined by the appended claims. [Brief explanation of the drawing]

[0017] [Figure 1] This figure shows the determination of the melting temperature (Tm). When the double strands of the labeled oligonucleotide and its complementary target are heated, the RFU decreases as the strands dissociate from each other (see Example 1). The black dots indicate the melting temperature. A) Melting curve of the probe / target double strand. The melting temperature is the point where the slope is steepest. B) First negative derivative (-dF / dT) of the melting curve of A. The maximum value of the curve is the melting temperature. [Figure 2] This figure shows the ΔCt values ​​for methylated DNA (mC) and unmethylated DNA (non-mC). ΔCt is calculated as the difference between the Ct value of the reference assay and the Ct value of the methyl-specific assay for mC or non-mC DNA, respectively. Note that the black lines are explanatory for the calculation and do not represent the exact Ct values. [Figure 3]This figure illustrates the base primer approach. The base primer consists of an anchor (black), a loop (dark gray), and a starter sequence (light gray) containing a hydrophobic nucleotide(s) that targets a region potentially subject to epigenetic modification. A) If the template DNA is methylated, the anchor sequence has high affinity to the template, and the anchor and starter sequences of the base primer anneal to the template DNA, potentially causing extension. B) The base primer does not have high affinity to unmethylated templates and therefore will not bind to unmethylated template DNA under selected conditions. This ensures that unmethylated templates are not amplified. Note that the dark gray lines represent the template strand, the light gray represents the amplicon, and the black circles represent methylation sites. When the base primer acts as a primer, the loop and anchor sequences are incorporated into a template that may work for the reverse primer. Extension from a reverse primer using a template containing the base primer typically stops at the anchor sequence because most DNA polymerases cannot read the hydrophobic nucleotides. The loop sequence and starter sequence function as normal primers after methyl-specific amplification occurs (created with BioRender.com). [Figure 4] This figure shows bar graphs of the difference in melting temperatures (ΔTm) for different types of probes, each with a different design and containing hydrophobic nucleotides, when hybridizing to complementary methylated or unmethylated sequences. Probes MGMT_Z, MGMT_E, MGMT_E2, MGMT_E3, and MGMT_Ref (see sequences in Table 1) were individually mixed with either an unmethylated (MGMT_0mC) target or a fully methylated (MGMT_1,2,3,4mC) target (see sequences in Table 1). The difference in melting temperatures between fully methylated and unmethylated targets is shown. Three copies were prepared for each melting experiment. The average value is shown above the bar. [Figure 5]This figure shows the melting peaks (first-order negative derivatives of the melting curves) of probe MGMT_E and reference probe MGMT_Ref (see sequences in Table 1) hybridized to targets MGMT_1,2,3,4mC and MGMT_0mC, respectively. It was confirmed that MGMT_E has a high melting temperature when hybridized to both methylated and unmethylated targets, but at the same time, its ability to distinguish between them is improved (ΔTm is larger) compared to the reference probe. [Figure 6] This graph shows a correlation between the number of methylation sites under the probe and the affinity of the probe. A linear fit between the number of methylation sites and the melting temperature of probe MGMT_E is shown. The linear fit was applied to the melting temperatures of probe MGMT_E hybridized to target MGMT_1mC, MGMT_2mC, MGMT_3mC, MGMT_4mC, each having one methylated cytosine, as well as MGMT_2,3mC and MGMT_1,2,3,4mC (see sequences in Table 3), each having two and four methylated cytosines, respectively. Three copies were prepared for each melting experiment. [Figure 7] This figure shows the melting peak of probe MGMT_E in the AmpliQueen master mix (available from PentaBase). The first negative derivative of the melting curves of probe MGMT_E hybridized to target MGMT_1,2,3,4mC and MGMT_0mC, respectively, is also shown. The MGMT_E / MGMT_0mC double strand is confirmed to dissociate completely at 80°C. This suggests that at approximately 80°C, probe MGMT_E can selectively bind to the methylated complementary sequence in the master mix. [Figure 8]This figure shows bar graphs of the difference in PCR cycles for signal (ΔCt) between base primers and reference primers under different annealing times and temperatures. The difference in ΔCt between the methyl-specific assay and the reference assay, and between methylated and unmethylated DNA, is used to indicate the optimal annealing conditions. Annealing temperatures and times are shown in the figure. For the reference assay, 700nM MGMT_Fw2C and MGMT_Rev2C, and 500nM MGMT_Probe2E were used as described in the "Base Primer Design" section of the Examples below. For the methyl-specific assay, MGMT_FwLoop4C primer was used instead of MGMT_Fw2C. For experiments at 79°C, 1% ZUBR Green was used instead of MGMT_Probe2E. [Figure 9] This figure shows the PCR curves for reference and methyl-specific assays for methylated and unmethylated DNA. Relative fluorescence units (RFU) of the probes are plotted in relation to the cycles. PCR program E (see Table 10) was used. The samples tested were methylated DNA isolated from IDH U87 WT(mC) and DNA (unmC) obtained from a healthy donor as a control. 700 nM MGMT_Fw2C, 700 nM MGMT_Rev2C, and 1% ZUBR Green were used in the reference assay (Ref), while the methyl-specific assay used 700 nM MGMT_FwLoop4C instead of MGMT_Fw2C. [Figure 10]This figure shows dot plots of MGMT_FwLoop5C (see Table 12 for sequences) at different annealing times and temperatures. The effects of changes in annealing time and temperature are shown using the ΔCt between the methyl-specific assay and the reference assay, and the difference in ΔΔCt between methylated DNA and unmethylated DNA. A) Annealing time fixed at 30 seconds, temperature varied. B) Annealing temperature fixed at 81°C, annealing time varied. The reference assay contained 700 nM MGMT_Fw2C_suE and MGMT_Rev2C_suE and 1% ZUBR Green. The methyl-specific assay contained MGMT_FwLoop5C instead of MGMT_Fw2C_suE. [Figure 11] This figure shows PCR curves for reference assays and methyl-specific assays for methylated and unmethylated DNA. RFUs are plotted corresponding to cycles. PCR program F (see Table 15) was used. The reference assay used 700nM MGMT_Fw2C_suE, 700nM MGMT_Rev2C_suE, and 1% ZUBR Green, while the methyl-specific assay used 500nM MGMT_FwLoop5C instead of MGMT_Fw2C_suE. [Figure 12] This figure shows the linear results of the reference assay and the methyl-specific assay. Ct is plotted in correspondence with the logarithm of the template input amount [log(q)]. A) Results of the reference assay. B) Results of the methyl-specific assay using MGMT_FwLoop5C. PCR program F was used. The reference assay used 700nM MGMT_Fw2C_suE, 700nM MGMT_Rev2C_suE and 1% ZUBR Green, while the methyl-specific assay used 500nM MGMT_FwLoop5C instead of MGMT_Fw2C_suE. [Figure 13]This figure shows the sensitivity test results using the MyGo Pro real-time PCR instrument (It-Islifescience). ΔCt is plotted in relation to the methylation rate in percent. PCR program F (see Table 15) was used. The reference assay used 700nM MGMT_Fw2C_suE, 700nM Rev2C, and 1% ZUBR Green, while the methyl-specific assay used 500nM MGMT_FwLoop5C instead of MGMT_Fw2C_suE. [Figure 14] This figure shows bar graphs of Tm and ΔTm for different types of mutant probes (probes with mismatches). MGMT probes containing hydrophobic nucleotides of type E (E_M1, E_M2, E_M3, E_M4, E_M5) and MGMT probes without (E) mutations (mismatches) were individually mixed with unmethylated (0mC) or fully methylated (1,2,3,4mC) targets, respectively. The tested MGMT probes and MGMT target sequences are shown in Table 20. The difference in melting temperature between methylated and unmethylated targets is shown. The mean value is shown above the bar. [Figure 15] This figure shows sensitivity tests performed on a BaseTyper® real-time PCR instrument using base primers with mismatches in the anchor sequence. A) ΔCt is plotted in relation to the methylation rate %. B) ΔCt is plotted in relation to log(methylation rate %). PCR program G (see Table 23) was used. 700nMMGMT_Fw2C, 700nMRev2E, and 500nMMGMT_Probe2E were used in the reference assay, and 700nMMGMT_FwLoop5D_M5 was used instead of MGMT_Fw2C in the methyl-specific assay. The template was artificial DNA from the MGMT promoter region methylated using CpG methyltransferase (New England Biolabs Inc, Ipswich, MA, USA). Linearity was observed in B), indicating that the assay is quantitative. [Figure 16]This figure shows base primers with a nucleotide gap between the 3' end of the anchor sequence (An) on the complementary target sequence and the 5' end of the starter sequence (St). Base primers with 0, 2, and 4 nucleotide gaps are shown (created with BioRender.com). [Figure 17] This figure shows bar graphs of ΔCt for base primers with mismatches in the anchor sequence. The base primers were designed to have a nucleotide gap between the 3' end of the anchor sequence on the complementary target sequence and the 5' end of the starter sequence. PCR program H (see Table 25) was used. For the reference assay, 700nM MGMT_Fw2C, 700nM MGMT_Rev2E, and 500nM MGMT_Probe2E were used, and for the methyl-specific assay, the 700nM base primers listed in Table 24 were used instead of MGMT_Fw2C. ΔCt is plotted for 100% methylation (100%mC), 10% methylation (10%mC), and unmethylated DNA (non.mC). The template was artificial DNA from the MGMT promoter region methylated using CpG methyltransferase (New England Biolabs Inc, Ipswich, MA, USA). [Figure 18] This diagram illustrates the design approach for a co-primer complex. The co-primer complex consists of two primers. One primer (dark gray), called the co-primer, contains an anchor sequence complementary to the target DNA and a stem sequence not complementary to the target sequence. The stem sequence is complementary to the sequence of the second primer. The second primer has a sequence complementary to the target DNA at its 3' end. Thus, the two primers form a tertiary complex. A) Under selected conditions, the complex is stable only when the template DNA is methylated. The second primer of the co-primer complex is used for the remainder of the PCR cycle. B) If the template DNA is not methylated, the co-primer complex is unstable under selected conditions and amplification does not occur (created with BioRender.com). [Figure 19]This figure shows the PCR curves of the auxiliary primer complex. Relative fluorescence units (RFU) are plotted in relation to the cycles. PCR program H (see Table 25) was used. For the reference assay, 700nM MGMT_Fw2C, 700nM MGMT_Rev2E, and 500nM MGMT_Probe2E were used, and for the methyl-specific assay, 700nM MGMT_Primer_1D and 50nM MGMT_Assist_1A_E were used instead of MGMT_Fw2C. The templates were the MGMT promoter region (non.mC) and artificial DNA from the same template (mC) methylated using CpG methyltransferase (New England Biolabs Inc, Ipswich, MA, USA). [Modes for carrying out the invention]

[0018] definition The term "anchor sequence" refers to a sequence contained within an oligonucleotide, preferably within a base primer. An anchor sequence contains one or more hydrophobic nucleotides located adjacent to a nucleotide complementary to the nucleotide of interest (NOI). Furthermore, an anchor sequence can hybridize to a portion of the target nucleic acid sequence, called the "target anchor sequence." The term "anchor sequence" is abbreviated as "An" herein.

[0019] As used herein, the term “base primer” refers to an oligonucleotide comprising, or consisting of, an anchor sequence (An), a loop sequence (Lp), and a starter sequence (St). Base primers are described in more detail below. The anchor sequence and starter sequence are at least partially complementary to the target nucleic acid sequence and can therefore hybridize to it, while the loop sequence is not complementary to the target nucleic acid sequence. Typically, neither the loop sequence nor the starter sequence contains hydrophobic nucleotides. If a base primer contains hydrophobic nucleotides between the anchor sequence and the loop sequence, such hydrophobic nucleotides are considered to be part of the anchor sequence. The terms “loop sequence” and “protruding sequence” are used interchangeably herein.

[0020] As used herein, the term “complementary” refers to a sequence of nucleotides that can be base-paired with another sequence of nucleotides by Watson-Crick base pairing.

[0021] As used herein, the term “hydrophobic nucleotide” refers to the hydrophobic nucleotides described in detail in the following section, “Hydrophobic Nucleotides.” In particular, the hydrophobic nucleotides according to the present invention contain intercalators linked to nucleotide / nucleotide analog / skeletal monomer units via linkers.

[0022] As used herein, the term “melting temperature” refers to the temperature at which 50% of a nucleic acid sequence is hybridized, relative to the unhybridized form capable of forming a double helix, and expressed in Celsius. The melting temperature is (T m It may also be called ). Nucleic acid melting refers to the thermal separation of the two strands of a double-stranded nucleic acid molecule. The melting temperature is preferably determined as described in Example 1 below. The melting temperature is preferably determined in the same solution (e.g., buffer) used to carry out the method of the present invention.

[0023] The terms "nucleotide methylation" or "methylated nucleotide" refer to the covalent modification of a nucleotide's nucleic acid base by an additional methyl group compared to the base normally present in the nucleic acid (in nucleic acids without epigenetic modification). While different methylations are relevant to this invention, the preferred methylation is cytosine methylation. Often, the methyl group is added to the fifth atom of the pyrimidine ring, creating 5-methylcytosine (5-mC).

[0024] [ka]

[0025] Cytosine methylation is particularly common in CpG dinucleotides, sometimes called "CpG sites," when cytosine is positioned immediately before guanine. Certain regions of the genome contain numerous CpG sites. Such regions are sometimes called "CpG islands."

[0026] As used herein, the term “nucleotide” refers to nucleotides, such as naturally occurring ribonucleotides or deoxyribonucleotides, or naturally occurring derivatives of ribonucleotides or deoxyribonucleotides. Examples of nucleotides include deoxyribonucleotides containing one of four nucleic acid bases: adenine (A), thymine (T), guanine (G), or cytosine (C), and ribonucleotides containing one of four nucleic acid bases: adenine (A), uracil (U), guanine (G), or cytosine (C). For brevity, nucleotides containing a particular nucleic acid base may be referred to herein simply by the name of the nucleic acid base. For example, a nucleotide containing cytosine (C) may be referred to as “cytosine” or “C”.

[0027] As used herein, the term “target nucleotide” refers to a nucleotide whose methylation status is to be investigated. The target nucleotide may be any nucleotide, but is preferably cytosine (C). “Target nucleotide” is abbreviated herein as “NOI”.

[0028] As used herein, the term “oligonucleotide” refers to a nucleotide and / or a nucleotide analog and / or an oligomer of a hydrophobic nucleotide. Preferably, an oligonucleotide is an oligomer of a nucleotide comprising optionally one or more hydrophobic nucleotides.

[0029] The term "target nucleic acid sequence" refers to a nucleic acid sequence containing a "target anchor sequence," which contains at least one NOI.

[0030] The term "target anchor sequence" refers to the portion of the target nucleic acid sequence containing the NOI. The "anchor sequence" can hybridize to the target anchor sequence.

[0031] The term "epigenetic modification" refers to modifications that can occur naturally in nucleic acid bases, taking into account that such modifications alter the stacking efficiency of nucleic acid bases.

[0032] As used herein, the term “epigenetic modification” typically refers to covalent modifications of nucleic acid bases that are inherited by daughter cells. Epigenetic modifications are preferably methylation, but may be alkylation, acetylation, hydroxylation, methoxylation, or other modifications of nucleic acid bases.

[0033] As used herein, the term “unmodified” means that the nucleic acid of interest has not undergone any modification or pretreatment step to convert unmethylated nucleotides to detectable moieties and / or methylated nucleotides to detectable moieties. Examples of such modifications or pretreatments include bisulfite conversion, restriction enzyme digestion, or TET enzyme conversion. Thus, unmodified as defined herein means only the absence of nucleic acid treatment or modification that selectively acts on either methylated or unmethylated nucleic acids. Pretreatment steps or modifications that do not selectively act on or distinguish either methylated or unmethylated nucleic acids, such as dilution, DNA purification, or enzymatic treatment that do not distinguish between methylated and unmethylated nucleic acids, are not included in this term as used herein.

[0034] Methylation status of the target nucleotide in the target nucleic acid sequence The present invention relates to a method for determining the epigenetic (such as methylation) state of at least one NOI in a target nucleic acid sequence.

[0035] This method is based on the use of oligonucleotides containing an anchor sequence, the anchor sequence comprising one or more hydrophobic nucleotides. The oligonucleotide may be any of the oligonucleotides described in the following section of this specification, “Oligocytes Containing Anchorage Sequences.” Oligonucleotides containing an anchor sequence have a significantly different affinity for nucleic acid sequences with epigenetic modifications (e.g., methylation) than for nucleic acid sequences without epigenetic modifications. For example, oligonucleotides containing an anchor sequence have a significantly higher affinity for methylated nucleic acids compared to unmethylated nucleic acids. This difference in affinity can be used to detect epigenetic modifications such as methylation in various ways, as described below.

[0036] Therefore, the method of the present invention generally includes step b) incubating the oligonucleotide with the target nucleic acid of interest at a temperature higher than the melting temperature between the oligonucleotide and the target nucleic acid sequence of interest, if the NOI has an epigenetic modification pattern different from the pattern being investigated.

[0037] Preferably, the temperature is selected to be higher than the melting temperature between the oligonucleotide and the target nucleic acid sequence when the NOI is not methylated, and the temperature is at most 2°C higher, equal to, or lower than the melting temperature between the oligonucleotide and the target nucleic acid sequence when the NOI is methylated.

[0038] In some embodiments, the temperature is selected to be at least 2°C higher than the melting temperature between the oligonucleotide and the target nucleic acid sequence when the NOI is unmethylated, and the temperature is up to 2°C higher, equal to, or lower than the melting temperature between the oligonucleotide and the target nucleic acid sequence when the NOI is methylated.

[0039] As described elsewhere in this specification, at the melting temperature, approximately 50% of the material exists in hybridized and non-hybridized forms. Therefore, at temperatures above the melting temperature, some hybridization typically still occurs. Thus, temperatures slightly above the melting temperature, for example up to 2°C, can be used.

[0040] Differences in affinity, and therefore epigenetic states such as methylation, can be detected using a variety of different methods. In very simple terms, the melting temperature between an oligonucleotide containing an anchor sequence and a target nucleic acid sequence can be determined, and based on this, the epigenetic state can be determined.

[0041] Preferably, the method includes an amplification step, which is designed so that amplification occurs only if the NOI is methylated, or only if the NOI is not methylated. This can be achieved by designing an assay that includes one or more steps, using a temperature that allows annealing of an oligonucleotide containing an anchor sequence to a methylated target nucleic acid, but does not allow annealing of the oligonucleotide to a non-methylated target sequence.

[0042] A preferred method is a PCR-based method, and in particular any of the PCR-based methods described in the following section “PCR” of this specification.

[0043] As described above, one advantage of the method of the present invention is that it does not require pretreatment of the target nucleic acid to convert unmethylated or methylated nucleotides to a detectable portion, such as bisulfite conversion, restriction enzyme digestion, or TET enzyme conversion.

[0044] Bisulfite converts unmethylated cytosine to uracil. 5-methylcytosine remains unchanged. This creates a difference in base pair sequences depending on the methylation state, allowing for the detection of differences between methylated and unmethylated DNA by methods such as PCR.

[0045] The methylation status of a sample can also be determined using methylation-sensitive restriction enzymes. Typically, methylation-sensitive restriction enzymes exist together with methylation-insensitive isoschisomers. An example of such an enzyme set is HpaII and MspI, both of which recognize the CCGG sequence and cleave before the CG dinucleotide. HpaII cleaves only unmethylated CCGG, while MspI cleaves both methylated and unmethylated CCGG sites. This allows for the detection of differences in digestion between methylated and unmethylated DNA, which can then be analyzed by PCR.

[0046] TET enzyme conversion relies on two enzymes, APOBEC and TET2. TET2 converts 5-methylcytosine to 5-carboxycytosine. This conversion prevents 5-methylcytosine from being converted by APOBEC, thereby deaminating only unmethylated cytosine to uracil, which allows for the detection of the difference between methylated and unmethylated DNA using methods such as PCR.

[0047] In some embodiments, the target nucleic acid sequence of interest is unmodified. Therefore, the absence of a step in the method of pretreatment of the nucleic acid of interest to convert unmethylated nucleotides to detectable moieties and / or methylated nucleotides to detectable moieties is included in the present invention. In some embodiments, the target nucleic acid sequence of interest has not undergone any treatment including bisulfite conversion, restriction enzyme digestion, or TET enzyme conversion before carrying out the method disclosed herein. In particular, it is preferable that the method does not include the bisulfite conversion step.

[0048] Target nucleic acid sequence The target nucleic acid sequence can be any nucleic acid sequence containing at least one nucleotide (NOI) whose epigenetics, such as methylation status, are desirable to determine. Typically, the target nucleic acid sequence is a longer nucleic acid sequence containing the desired short sequence, and is referred to herein as the “target anchor sequence.” The target anchor sequence contains at least one NOI.

[0049] Preferably, the nucleic acid is DNA, but the nucleic acid may be other types of nucleic acids such as RNA. In a preferred embodiment, the nucleic acid is genomic DNA. This method can be advantageously used to determine epigenetic modifications of naturally occurring sequences, particularly cytosine methylation.

[0050] Preferably, the epigenetic modification is methylation, but may be alkylation, acetylation, hydroxylation, methoxylation, or other modifications of the nucleic acid base.

[0051] The target anchor sequence contains one or more NOIs. The anchor sequence of the oligonucleotide of the present invention is selected in such a manner that the anchor sequence can hybridize with the target anchor sequence. Typically, the anchor sequence is at least 50% complementary to the target anchor sequence.

[0052] In many cases, several NOIs, which may or may not be modified (e.g., methylated), are located in close proximity within the target nucleic acid sequence. In such cases, it may be desirable to determine the overall modification (methylation) state of all nucleotides. This can be done by the method of the present invention described below, using oligonucleotides having an anchor sequence containing one hydrophobic nucleotide per NOI. The invention also includes determining the modification (methylation) state of only one or more NOIs. Again, this can be done by using oligonucleotides having an anchor sequence containing one hydrophobic nucleotide per NOI under investigation.

[0053] In some embodiments, the target nucleic acid sequence may be a nucleic acid sequence containing one or more NOIs whose epigenetic modification state is associated with a clinical condition. For example, the methylation state of an NOI may be associated with the presence of a clinical condition, the progression of a clinical condition, the risk of acquiring a clinical condition, or susceptibility to a particular treatment, as will be described in more detail in the "Clinical Conditions" section below. The "association" of an NOI methylation state with a clinical condition does not mean that it is the cause of that clinical condition, but rather that a given methylation state is more common with respect to that clinical condition.

[0054] NOI can be any nucleotide for which it is desirable to detect the epigenetic modification state. In a preferred embodiment, NOI is cytosine (C), and more preferably, NOI is cytosine located at the CpG site. In the most preferred embodiment, the epigenetic modification of NOI is methylation of the CpG site.

[0055] The target nucleic acid may be contained in any composition containing the target nucleic acid sequence. Often, the target nucleic acid is contained in a nucleic acid sample. The sample may be obtained from any source for which it is desirable to determine the epigenetic state (e.g., methylation state). For example, the sample may be obtained from a mammal such as a human. The sample may be at least partially purified; that is, the sample may contain at least partially purified nucleic acid. Therefore, the target nucleic acid sequence of interest may be contained in the DNA purified from the sample.

[0056] As described elsewhere in this specification, the target anchor sequence is at least partially complementary to the anchor sequence. Therefore, if the target nucleic acid is a double-stranded nucleic acid such as DNA, the anchor sequence is usually at least partially identical to one strand of DNA, and the target anchor sequence is located on the other strand of DNA.

[0057] Oligonucleotides containing anchor sequences The present invention relates to a method for determining epigenetics, such as the methylation state of at least one NOI, the method employing the use of an oligonucleotide comprising an anchor sequence (An), wherein the anchor sequence is a sequence at least 50% complementary to a site in a target nucleic acid sequence comprising the NOI, which is referred to herein as the “target anchor sequence”. The anchor sequence preferably comprises at least one hydrophobic nucleotide (H) located between the nucleotide complementary to the NOI and the nucleotide in the oligonucleotide immediately 5' to it.

[0058] In embodiments of the present invention where NOI is C, the anchor array therefore preferably includes the array-HG-, where G is complementary to the desired C.

[0059] The anchor sequence preferably does not contain two adjacent hydrophobic nucleotides. Therefore, the anchor sequence specifically includes the sequence -NHG-, where N is any nucleotide and the sequence -NG- is complementary to the cytosine of interest and its adjacent nucleotides. In particular, N can be selected from the group consisting of C, G, A, and T.

[0060] In embodiments where the NOI is C located at the CpG site, it is preferable that the anchor sequence includes the sequence -CHG-.

[0061] The anchor sequence may contain two or more hydrophobic nucleotides. Therefore, the anchor sequence may contain at least two, for example, at least three, for example, at least four, for example, in the range of 1 to 10, for example, in the range of 2 to 10, for example, in the range of 3 to 10, for example, in the range of 4 to 10 (H).

[0062] The anchor sequence may, in particular, contain one hydrophobic nucleotide per NOI. Preferably, all hydrophobic nucleotides are located between a nucleotide complementary to the NOI and a nucleotide in the oligonucleotide immediately 5' to it. Furthermore, the anchor sequence may also contain additional hydrophobic nucleotides.

[0063] In embodiments of the present invention, when the target nucleic acid sequence contains two or more NOIs which are C, the anchor sequence may contain two or more -NHG- sequences, where each N can individually be any nucleotide. Preferably, the anchor sequence contains one -NHG- sequence per NOI which is C, and each -NG- sequence is complementary to the cytosine of interest and its adjacent nucleotides. Thus, the anchor sequence may contain at least two, for example, at least three, for example, at least four, for example, in the range of 2-5, for example, in the range of 3-5, for example, in the range of 4-5, where each N is individually any nucleotide, and each -NG- sequence is complementary to the cytosine of interest and its adjacent nucleotides. In particular, N can be selected from the group consisting of C, G, A, and T.

[0064] In an embodiment of the present invention, when the target nucleic acid sequence contains two or more NOIs that are C located at the CpG site, the anchor sequence can contain one -C-H-G- sequence per NOI that is C located at the CpG site. Therefore, the anchor sequence can contain at least two, for example at least three, for example at least four, for example in the range of 2 to 5, for example in the range of 3 to 5, for example in the range of 4 to 5 -C-H-G- sequences.

[0065] In one embodiment, the oligonucleotide contains an anchor sequence having the following general structure: 5’(N) n -HG-(N) m -HG-(N) p -3’ (where N is any nucleotide or nucleotide analog; G is the nucleotide guanine; n is an integer of 0 or more; m is an integer of 1 or more; p is an integer of 0 or more; H is a hydrophobic nucleotide, for example any of the hydrophobic nucleotides described below).

[0066] In one embodiment, the oligonucleotide contains an anchor sequence having the following general structure: 5’(N) n -HG-(N) m -HG-(N) p -HG-(N) q -3’ (where p is 1 or more; [[ID=XX]] q is an integer of 0 or more).

[0067] In one embodiment, the oligonucleotide contains an anchor sequence having the following general structure: 5’(N) n -YG-(N) m -YG-(N) p -YG-(N) q -YG-(N) It should be noted that there seems to be an error in the original text where "XX" is marked in the ID=XX line. This might be a typo in the original. The translation is done based on the provided text as accurately as possible.u -3' (In the formula, q is an integer greater than or equal to 1; (where u is a non-negative integer).

[0068] Y represents C or T according to the extended IUPAC code.

[0069] Regarding the oligonucleotides described above, n may be an integer in the range of 0 to 4, for example, or n may be 1, m may be an integer in the range of 1 to 8, for example, or 1 to 6, p may be an integer in the range of 1 to 50, for example, 1 to 30, for example, 1 to 10, for example, or 1 to 6, q may be an integer in the range of 1 to 50, for example, 1 to 30, for example, 1 to 10, for example, or 1 to 6, and u may be an integer in the range of 1 to 50, for example, 1 to 30, for example, 1 to 10, for example, or 1 to 6.

[0070] In preferred embodiments of the present invention, the oligonucleotide is a base primer as described below. In such embodiments, the anchor sequence is linked to a loop sequence and a starter sequence, and in such embodiments, the 3' portion of the oligonucleotide is long enough to contain both the loop sequence and the starter sequence.

[0071] As explained above, NOI can be a cytosine located at a CpG site. In such cases, it is preferable that at least one nucleotide immediately 5' to the H is a cytosine (C). If all NOIs are cytosines located at a CpG site, and the H is adjacent to a G complementary to the NOI, it is preferable that all nucleotides immediately 5' to the H are cytosine (C).

[0072] Oligonucleotides containing an anchor sequence anneal to methylated target nucleic acid sequences with higher affinity than unmethylated target nucleic acid sequences. Therefore, the melting temperature between the oligonucleotide and the target methylated target nucleic acid sequence is often at least 5°C higher, for example at least 6°C higher, for example in the range of 6–15°C higher, for example in the range of 6–12°C higher, than the melting temperature between the oligonucleotide and the target unmethylated target nucleic acid sequence.

[0073] In particular, the present invention is based on the discovery that the oligonucleotides described herein have a greater difference in affinity for methylated versus unmethylated target nucleic acids compared to similar oligonucleotides that do not contain hydrophobic nucleotides. This large difference in affinity allows for successful detection of methylation by simple methods such as PCR. The difference in melting temperature between the oligonucleotide of the present invention and the methylated target nucleic acid sequence compared to the target unmethylated target nucleic acid sequence is preferably at least 1°C higher than the difference in melting temperature between an oligonucleotide with the same sequence as the unmethylated target nucleic acid sequence, except that it lacks hydrophobic nucleotides, and the methylated target nucleic acid sequence.

[0074] NOI methylation, such as cytosine methylation, i.e., the presence of methylated cytosines (or more) in nucleic acids, such as the target nucleic acid, causes a difference in the melting temperature when the nucleic acid hybridizes with an oligonucleotide. This difference depends on the number of methylated NOIs and can be difficult to detect. The hydrophobic nucleotides in the oligonucleotides of the present invention amplify the difference in affinity between unmethylated and methylated NOIs compared to oligonucleotides that are otherwise identical but do not contain hydrophobic nucleotides. Therefore, depending on the number of methylated NOIs such as methylated cytosines, the oligonucleotides of the present invention result in a larger difference in melting temperature when hybridized to the target sequence compared to identical oligonucleotides that do not contain hydrophobic nucleotides, thus facilitating the detection of the presence of methylated NOIs.

[0075] In some embodiments, A. i. When hybridized to a target nucleic acid sequence, the oligonucleotide of the present invention contains hydrophobic nucleotides (multiple nucleotides), wherein the target nucleic acid sequence contains at least one methylated NOI, such as at least one methylated cytosine; ii. The oligonucleotide of the present invention, which contains hydrophobic nucleotides (or more) when hybridized to a target nucleic acid sequence, wherein the target nucleic acid sequence does not contain methylated NOIs such as methylated cytosine; The absolute difference in melting temperatures, and B. An oligonucleotide identical to iA, but which, when hybridized to the target nucleic acid sequence, does not contain hydrophobic nucleotides (multiple nucleotides are possible), and whose target nucleic acid sequence contains the same number of methylated NOIs as Ai, for example, the same number of methylated cytosines as Ai; ii.A. The oligonucleotide is the same as the oligonucleotide in ii.A., but when hybridized to the target nucleic acid sequence, it does not contain hydrophobic nucleotides (multiple nucleotides are possible), and the target nucleic acid sequence does not contain methylated NOIs such as methylated cytosine; The absolute difference in melting temperatures is For each NOI such as cytosine that is methylated instead of unmethylated in the target nucleic acid sequence of interest, the difference is at least 0.50°C, for example at least 0.75°C, preferably at least 1.0°C, where the difference is AB and the difference is positive. In a preferred embodiment, the melting temperature is measured in TM buffer containing 0.02 M Na2HPO4, 0.02 M NaCl, and 2 mM EDTA.

[0076] In some embodiments, i. The oligonucleotide of the present invention, which, when hybridized to a target nucleic acid sequence, contains a hydrophobic nucleotide, wherein the target nucleic acid sequence contains at least one methylated NOI, such as at least one methylated cytosine; ii. When hybridized to a target nucleic acid sequence, the oligonucleotide of the present invention contains a hydrophobic nucleotide, and the target nucleic acid sequence does not contain methylated NOIs such as methylated cytosine; The absolute difference between the melting temperatures is, For each NOI such as cytosine, the melting temperature is at least 1.6°C, for example at least 1.8°C, for example at least 2.0°C, for example at least 2.2°C, preferably at least 2.4°C, where the target nucleic acid sequence is methylated instead of not methylated. In a preferred embodiment, the melting temperature is measured in TM buffer containing 0.02 M Na2HPO4, 0.02 M NaCl, and 2 mM EDTA.

[0077] For comparison, in some embodiments, i. An oligonucleotide identical to the oligonucleotide described in the paragraph above, but which, when hybridized to the target nucleic acid sequence, does not contain hydrophobic nucleotides, and the target nucleic acid sequence contains at least one methylated NOI, such as at least one methylated cytosine; ii. An oligonucleotide identical to the oligonucleotide described in the paragraph above, but which, when hybridized to the target nucleic acid sequence, does not contain hydrophobic nucleotides, and the target nucleic acid sequence does not contain methylated NOIs such as methylated cytosine; The absolute difference in melting temperatures is For each NOI such as cytosine that is methylated instead of unmethylated in the target nucleic acid sequence of interest, the melting temperature is up to 1.0°C, e.g., up to 0.9°C, e.g., up to 0.8°C. In preferred embodiments, the melting temperature is measured in TM buffer containing 0.02 M Na2HPO4, 0.02 M NaCl, and 2 mM EDTA.

[0078] Preferably, the anchor sequence (An) consists of nucleotides in the range of 8 to 30, for example, 8 to 20, for example, 10 to 20, for example, 15 to 20, and one or more of the nucleotides are hydrophobic nucleotides.

[0079] As mentioned, the anchor sequence (An) is at least 50% complementary to the target anchor sequence, and preferably at least 85% complementary to the target anchor sequence. In some embodiments, the anchor sequence may be 100% complementary to the target anchor sequence. However, in preferred embodiments, the anchor sequence is not 100% complementary to the target anchor sequence.

[0080] Base primer The present invention relates to a method for determining the epigenetic state, such as the methylation state, of at least one NOI, and the method utilizes the use of an oligonucleotide containing an anchor sequence (An). In preferred embodiments, the oligonucleotide is the base primer described in this section.

[0081] The base primer according to the present invention is an oligonucleotide having the following structure; 5'-An-Lp-St-3' In the formula, An is the anchor array; Lp is a loop array; St is the starter sequence.

[0082] The anchor sequence (An) is preferably a sequence that is at least 50% complementary to a site in the target nucleic acid sequence containing the NOI, which is referred to herein as the “target anchor sequence.” The anchor sequence preferably includes at least one hydrophobic nucleotide (H) located between the nucleotide complementary to the NOI and the nucleotide in the oligonucleotide immediately 5' to its right. The anchor sequence may be any of the anchor sequences described in particular in the above section, “Oligoligonucleotides containing anchor sequences.”

[0083] A loop sequence (Lp) is a sequence that does not hybridize to the target nucleic acid sequence to any significant degree. Therefore, preferably, the Lp is not complementary to the nucleic acid sequence of interest. The loop sequence may consist of a single nucleic acid sequence capable of forming a protruding structure, or of two or more nucleic acid sequences that can hybridize to each other at least partially to form a complex capable of forming a protruding structure. In some embodiments, the protruding structure is a loop structure. In some embodiments, the protruding structure is a stem structure.

[0084] In some embodiments, the oligonucleotide of the present invention may thus consist of a first and a second nucleic acid sequence, the first nucleic acid sequence comprising an anchor sequence (An) complementary to the target DNA and a first portion of a loop sequence not complementary to the target nucleic acid sequence of choice, the second nucleic acid sequence comprising a second portion of a loop sequence that can at least partially hybridize to the first portion of the loop sequence, and the second nucleic acid sequence further comprising a starter sequence (St). Upon hybridization, the first and second nucleic acids may form a protruding structure such as a stem structure.

[0085] In some embodiments, the oligonucleotide of the present invention may consist of first, second, and third nucleic acid sequences, the first nucleic acid sequence comprising an anchor sequence (An) complementary to the target DNA and a first portion of a loop sequence not complementary to the target nucleic acid sequence of choice, the second nucleic acid sequence comprising a second portion of a loop sequence that can at least partially hybridize to the first portion of the loop sequence, the second nucleic acid sequence further comprising a starter sequence (St), and the third nucleic acid sequence may hybridize to a portion of the 3' end of the first oligonucleotide in the first region and to a portion of the 5' end of the second oligonucleotide in the second region. Upon hybridization, the first, second, and third nucleic acids can form protruding structures such as loop structures.

[0086] In some embodiments, the oligonucleotide of the present invention is encoded as a single oligonucleotide. In some embodiments, the oligonucleotide of the present invention is encoded as two or more distinct oligonucleotides.

[0087] The starter sequence (St) is a sequence that can hybridize to the target nucleic acid sequence, but with low affinity. Therefore, under the conditions used in the method of the present invention, it is preferable that the starter sequence does not anneal to the target nucleic acid sequence unless the anchor sequence also anneals. The starter sequence (St) is preferably extensible, and therefore can act as a primer. Therefore, the starter sequence is preferably at least 90% complementary to a site of the target nucleic acid sequence, which is referred to herein as the "target starter sequence".

[0088] The target starter sequence is the target nucleic acid sequence located 5' to the target anchor sequence. The target starter sequence may be located immediately 5' to the target anchor sequence, but there may be space between the sequences. Therefore, the target starter sequence can be separated from the target anchor sequence within a range of 1–20 nucleotides, e.g., 2–10 nucleotides, e.g., 2–5 nucleotides.

[0089] As described above, the starter sequence is at least 90% complementary to the target starter sequence, but may also be 100% complementary to the target starter sequence.

[0090] Base primers are particularly useful in PCR-based methods, as they are one primer in a primer pair that can be used to amplify a target nucleic acid sequence. Based on the design of the anchor sequence, the anchor sequence anneals with high affinity to the methylated target nucleic acid sequence, thereby also enabling the annealing of the starter sequence. After annealing, the base primer can be extended using a polymerase, such as any DNA polymerase conventionally used in PCR. The first reverse transcription typically terminates when the polymerase encounters the first hydrophobic nucleotide in the product, and thus at least a portion of the anchor sequence is deleted. However, the base primer can still be used as a primer in subsequent PCR rounds because the loop sequence and starter sequence can anneal to the product of the first amplification round.

[0091] Polymerase typically stops elongating at a position where the template contains hydrophobic nucleotides; therefore, it may be preferable for the base primer to contain hydrophobic nucleotides between the anchor sequence and the loop sequence. Hydrophobic nucleotides, for example, are considered part of the anchor sequence.

[0092] Therefore, it is preferable that both the starter sequence and the loop sequence do not contain hydrophobic nucleotides. However, if the loop sequence consists of two or more nucleic acid sequences, the nucleic acid sequences (or more) not covalently linked to the starter sequence may contain one or more hydrophobic nucleotides.

[0093] The loop sequence and starter sequence function as primers for the first amplified round from the target. Therefore, the loop sequence and starter sequence (-Lp-St-) should be designed in such a way that they can function as primers. Accordingly, it is preferable that the oligonucleotide consisting of Lp-St has a melting temperature of at least 50°C, e.g., at least 55°C, e.g., at least 65°C, relative to its complementary sequence. In general, it is preferable that the loop sequence and starter sequence together consist of at least 15 nucleotides, e.g., at least 17 nucleotides, e.g., in the range of 15-45 nucleotides, e.g., in the range of 17-35 nucleotides, e.g., in the range of 17-25 nucleotides.

[0094] As described above, the starter sequence anneals to the target nucleic acid sequence with low affinity. Therefore, it is preferable that the starter sequence is not too long. Preferably, the starter sequence consists of nucleotides in the range of 5 to 15, for example, in the range of 8 to 14, or for example, in the range of 10 to 12.

[0095] The base primer may include additional components in addition to the anchor, loop, and starter sequences. For example, the base primer may be coupled to a detectable label. However, in a preferred embodiment, the base primer consists of the anchor, loop, and starter sequences.

[0096] Other oligonucleotides While preferred oligonucleotides according to the present invention are described above, in one embodiment, the present invention provides for example, T at a maximum temperature of 3°C, for example, at a maximum temperature of 2°C. m We also provide oligonucleotides that have similar affinity to methylated and unmethylated target nucleic acid sequences, such as differences in the methylation and unmethylation of the target nucleic acid sequence. When the NOI is a cytosine located at a CpG site, such oligonucleotides may include, or consist of, the following general structure: 5'(N) n -CGH-(N) m -CGH-(N) p -3' (wherein N is any nucleotide or nucleotide analogue; C is the nucleotide cytosine; G is the nucleotide guanine; n is a non-negative integer; m is an integer greater than or equal to 1; p is a non-negative integer; H is a hydrophobic nucleotide, Here, the hydrophobic nucleotide has the following structure; XYQ During the ceremony, X, Q, and Y may be as described in the “Hydrophobic Nucleotides” section of this specification.

[0097] Oligonucleotides may have, in particular, the following general structures: 5'(N) n -CGH-(N) m -CGH-(N) p -CGH-(N) q -3' (In the formula, p is greater than or equal to 1; q is a non-negative integer.

[0098] In one embodiment, such an oligonucleotide includes or consists of the following general structure: 5'(N) n -CGH-(N) m -CGH-(N) p -CGH-(N) q -CGH-(N) u -3' (In the formula, q is an integer greater than or equal to 1; (where u is a non-negative integer).

[0099] n, m, p, q, and u may be, for example, those described in the above section “Oligononucleotides Containing Anchor Sequences” of this specification.

[0100] Hydrophobic nucleotides The oligonucleotide used in the present invention comprises one or more hydrophobic nucleotides. The hydrophobic nucleotide according to the present invention has the following structure: XYQ (In the formula, X is a nucleotide or nucleotide analogue, or a skeletal monomer unit that can be incorporated into the backbone of a nucleic acid. Q is an intercalator that does not participate in Watson-Crick hydrogen bonding; Y is a linker portion that connects a nucleotide or nucleotide analog or backbone monomer unit to an intercalator.

[0101] Intercalator Q can be any intercalator. The term intercalator according to the present invention encompasses any molecular portion comprising at least one essentially flat conjugated system that can co-stacking with nucleic acid bases of a nucleic acid. Preferably, the intercalator according to the present invention consists essentially of at least one essentially flat conjugated system that can co-stacking with nucleic acid bases of a nucleic acid.

[0102] An intercalator contains at least one π (phi) electron system and, according to the present invention, can interact with other molecules containing π electron systems. These interactions can contribute positively or negatively to the hydrophobic interactions of the intercalator. Hunter and Sanders (1990) J.Am Chem.Soc.112:5525-5534 proposed various directions and conditions in which two π electron systems can positively interact with each other.

[0103] The intercalator may be any of the intercalators described in section “Intercalators” on pages 30, l.26 to 40, l.3 of the international patent application WO2017 / 045689. For example, the intercalator may have any of the structures described on pages 32-39 of the same international patent application WO2017 / 045689, the details of which are incorporated herein by reference.

[0104] In one embodiment, at least one Q, such as all intercalators, is selected from the group consisting of polyaromates and heteropolyaromates optionally substituted with one or more selected from the group consisting of hydroxyl, bromo, fluoro, chloro, iodo, mercapto, thio, cyano, alkylthio, heterocyclic, aryl, heteroaryl, carboxyl, carboxyl, carboxyl, alkyl, alkenyl, alkynyl, nitro, amino, alkoxyl, and amide. The polyaromate or heteropolyaromate may consist of at least three rings, for example, four rings.

[0105] The intercalator may be selected from the group consisting of, for example, benzene, pentalene, indene, naphthalene, azulene, as-indacene, s-indacene, biphenylene, acenaphthylene, phenalene, heptalene, phenanthrane, fluoranthene, phenanthroline, phenazine, phenanthridine, anthraquinone, pyrene, anthracene, napthene, phenanthrene, flurene, picene, chrysene, naphtacene, acridone, benzoanthracene, stilbene, oxalopyridocarbazole, azidobenzene, porphyrin, and psoralen, as well as their derivatives.

[0106] In one embodiment, at least one Q, such as all intercalators, comprises or consists of pyrene or pyrido[3',2':4,5]thieno[3,2-d]pyrimidine-4(1H)-one or 7,9-dimethylpyrido[3',2',4,5]thieno[3,2-d]pyrimidine-4(3H)-one. In particular, at least one of all intercalators may be pyrene.

[0107] The nucleotide or nucleotide analog backbone monomer unit according to the present invention is a part of the nucleotide involved in its incorporation into the backbone of the nucleic acid or nucleic acid analog. Preferably, the backbone monomer unit (X) is covalently linked to a linker (Y) which is covalently linked to an intercalator. Any suitable backbone monomer unit can be used to incorporate an intercalator into the oligonucleotide analog according to the present invention.

[0108] The skeletal monomer unit may be any of the skeletal monomer units described in international patent application WO2017 / 045689, section "Backbone monomer unit", pp. 40, l.5 to p. 56, l.3. The skeletal monomer unit may be any of the skeletal monomer units described in international patent application WO03 / 052132, section "Backbone monomer unit", pp. 24, l.27 to p. 43, l.14.

[0109] In a particularly preferred embodiment of the present invention, the hydrophobic nucleotide comprises a skeleton monomer unit containing a phosphoramidite, and more preferably, the skeleton monomer unit contains a trivalent phosphoramidite or a pentavalent phosphoramidite.

[0110] A suitable trivalent phosphoramidite is a trivalent or pentavalent phosphoramidite that can be incorporated into the backbone of nucleic acids and / or nucleic acid analogs. Typically, the amidite group itself cannot be incorporated into the nucleic acid backbone; rather, the amidite group or a portion of the amidite group can function as a leaving group and / or protecting group. However, it is preferable that the backbone monomer unit contains a phosphoramidite group, because such a group can facilitate the incorporation of the backbone monomer unit into the nucleic acid backbone.

[0111] The linker of the intercalator nucleotide according to the present invention is a portion that connects the intercalator to the backbone monomer of the hydrophobic nucleotide, and preferably links the intercalator and the backbone monomer unit by a covalent bond. The linker may include one or more atoms or interatomic bonds.

[0112] According to the definitions of skeleton and intercalator as defined above in this specification, a linker is the shortest path connecting the skeleton and the intercalator. If the intercalator is directly connected to the skeleton, the linker is a connection.

[0113] Linkers typically consist of a chain of atoms or a branched chain of atoms. The chains can be saturated or unsaturated. Linkers may also have a ring structure with or without conjugated bonds.

[0114] For example, the linker may include a chain of atoms selected from the group consisting of C, O, S, N, P, Se, Si, Ge, Sn, and Pb, preferably from the group consisting of C, O, S, N, and P, and more preferably C, with one end of the chain connected to an intercalator and the other end of the chain connected to a skeletal monomer unit.

[0115] The linker may be, for example, one of the linkers described in international patent application WO2017 / 045689, section "Linker", pp. 56, l. 5 to pp. 59, l. 10. The linker may also be one of the linkers described in WO03 / 052132, section "Linker", pp. 54, l. 15 to pp. 58, l. 7.

[0116] PCR The method of the present invention preferably includes the steps of (b) incubating the oligonucleotide of the present invention with the target nucleic acid of interest at a temperature higher than the melting temperature between the oligonucleotide and the target nucleic acid sequence of interest, if the NOI does not contain the epigenetic modification state to be investigated, for example, if the NOI is not methylated; and (c) detecting whether or not the oligonucleotide anneals to the target nucleic acid of interest.

[0117] These steps may, in a preferred embodiment, be carried out as part of a polymerase chain reaction (PCR). Preferably, the PCR is carried out such that most or all of the steps of the PCR are performed at a temperature higher than the melting temperature between the oligonucleotide and the target nucleic acid sequence of the choice, when the oligonucleotide does not contain the desired epigenetic modification state. More preferably, the PCR is carried out such that most or even all of the steps of the PCR are performed at a temperature higher than the melting temperature between the oligonucleotide and the target nucleic acid sequence of the choice when the NOI is not methylated, but at a temperature low enough to allow amplification between the oligonucleotide and the target nucleic acid sequence of the choice when the NOI is methylated.

[0118] PCR is typically, i. Melting temperature, ii. Annealing and Extension Temperature This includes multiple incubation cycles.

[0119] The annealing and extension temperatures may be the same or different.

[0120] Preferably, if the NOI is not methylated, PCR is performed in such a manner that the annealing and extension temperatures are higher than the melting temperature between the oligonucleotide and the target nucleic acid sequence in at least the first cycle, preferably one or more cycles of PCR, e.g., most cycles of PCR, e.g., all cycles of PCR. Furthermore, if the NOI is methylated, the temperature may be selected to be up to 2°C higher or lower than the melting temperature between the oligonucleotide and the target nucleic acid sequence.

[0121] The annealing temperature depends on the specific oligonucleotide, but can be in the range of, for example, 55-80°C.

[0122] In some embodiments of the present invention, the length and / or temperature of the PCR steps are modified during PCR. In particular, the first cycle, for example, the first 1 to 5 cycles, may be carried out in such a manner that methylated DNA is amplified in a more specific way. Thus, PCR may include one or more epigenetic modification-specific amplification cycles and one or more general amplification cycles, where the epigenetic modification-specific amplification cycle(s) are a. A step of dissolving nucleic acids, b. The step of annealing (and extending) under epigenetic modification-specific conditions, A typical amplification cycle is: a. A step of dissolving nucleic acids, b. A step of annealing (and stretching) under general conditions, including:

[0123] Melting typically involves incubation at a melting temperature of at least 90°C. Annealing and extension under epigenetic modification-specific conditions are usually carried out at a higher temperature than annealing and extension under general conditions, for example, at least 1°C higher, e.g., at least 2°C higher, than the temperature used for annealing and extension under general conditions. This method may include any suitable number of epigenetic modification-specific amplification cycles, e.g., in the range of 1 to 100 epigenetic modification-specific amplification cycles.

[0124] The step of incubation at the annealing (and extension) temperature can be carried out for any preferred time. For example, at least the first cycle of PCR may include incubation at the annealing temperature in the range of 5 to 120 seconds, e.g., in the range of 10 to 90 seconds. Annealing may also be carried out in the range of 30 to 90 seconds, e.g., in the range of 45 to 75 seconds, e.g., in the range of about 60 seconds.

[0125] PCR generally involves the use of a set of primers capable of amplifying a target nucleic acid sequence of interest. Preferably, one of the primers is an oligonucleotide according to the present invention, and more preferably, a base primer. If the forward primer is an oligonucleotide according to the present invention, e.g., a base primer, the reverse primer is typically a conventional primer, and vice versa.

[0126] Those skilled in the art can determine a useful concentration of the oligonucleotide of the present invention for use in PCR. For example, the concentration may be in the range of 300 to 700 nM, for example, about 500 nM.

[0127] PCR can be carried out in any preferred method known to those skilled in the art. In one embodiment, PCR is real-time PCR. Real-time PCR typically involves the use of a detectable label, such as a dye. In one embodiment, PCR, for example, real-time PCR, uses a probe containing a detectable label for the detection of the product. The probe may be any oligonucleotide capable of binding to the PCR product, but preferably the probe is an oligonucleotide capable of binding to the PCR product with at least the same affinity, and preferably with a higher affinity than each of the primers. Thus, the probe may consist of an oligonucleotide that is at least 90% complementary to the product of the PCR reaction and a detectable label. In a preferred embodiment, the probe contains one or more hydrophobic nucleotides, so that hydrophobic nucleotides increase the affinity of the probe.

[0128] If the PCR is real-time PCR, the PCR result can be determined as Ct. Therefore, if Ct is below a given threshold, the target nucleic acid of interest can be considered methylated. The threshold can be determined in advance or using a relevant control.

[0129] In addition to primers and probes, PCR includes PCR reagents. Those skilled in the art are well aware of the importance of selecting suitable PCR reagents. Generally, PCR reagents contain at least nucleotides and polymerase. Typically, PCR reagents also include suitable salts and buffers.

[0130] The polymerase can be any polymerase useful for PCR, for example, any DNA polymerase useful for PCR. In some embodiments, the polymerase is one that terminates its extension at any hydrophobic nucleotide in the template. This is the case for most conventional DNA polymerases used routinely in PCR.

[0131] Clinical condition The method of the present invention can be used to detect methylation of NOI, the methylation state of which is related to the clinical state.

[0132] Therefore, the method of the present invention may be used to determine whether an individual is at risk of suffering from a clinical condition, or the risk of an individual becoming clinical, where the clinical condition is related to the methylation status of NOIs such as cytosine in the nucleic acid of interest. Such a method is typically used, a. Steps to provide a sample from an individual, b. The method of the present invention includes the step of determining the methylation state of NOI in the above sample by carrying out the method of the present invention.

[0133] In some embodiments, a method is provided for determining whether an individual is at risk of having a clinical condition, or the risk of an individual developing a clinical condition, where the clinical condition is related to the methylation status of NOI in the nucleic acid of interest, and the method is a. To provide samples obtained from individuals; b. Determining the methylation status of NOI in a sample by performing the method of any one of the prior claims, wherein the methylation status indicates the presence or risk of developing a clinical condition.

[0134] The method of the present invention may also be used to determine the potential therapeutic effect of a clinical condition in an individual requiring it, where the therapeutic effect of the clinical condition is related to the methylation state of cytosine in the nucleic acid of interest. Such a method is typically used, i. Steps to provide a sample from an individual, ii. The method of the present invention includes the step of determining the methylation state of NOI in the above sample by carrying out the method of the present invention.

[0135] In some aspects of the present invention, a method is provided for determining the potential therapeutic effect of a clinical condition in an individual in need thereof, wherein the therapeutic effect of the clinical condition is related to the methylation state of NOI in the nucleic acid of interest, and the method is a. To provide samples obtained from individuals; b. Determining the methylation status of NOI in a sample by performing the methods disclosed herein, wherein the methylation status indicates the effect of different treatment regimens on a clinical condition.

[0136] In some embodiments, NOI is cytosine.

[0137] Numerous clinical conditions, including several types of cancer such as breast cancer, colon cancer, and liver cancer, as well as diseases such as rheumatoid arthritis and multiple sclerosis, have been shown to be associated with the methylation of one or more NOIs. Therefore, the methylation status of one or more NOIs may indicate the presence of a clinical condition, its progression, its severity, its prognosis, and the risk of acquiring it. Furthermore, the effectiveness of different treatment regimens has been shown to be associated with the methylation status of one or more NOIs. Thus, the method of the present invention can be used for all of the aforementioned detections. Clinical conditions may include, for example, cancer, such as glioblastoma.

[0138] In preferred embodiments, the methods disclosed herein above do not involve surgical steps.

[0139] In some embodiments, the methods disclosed herein are performed on samples isolated from a patient. In some embodiments, the sample is a bodily fluid sample. The bodily fluid sample may be blood, urine, sputum, breast milk, cerebrospinal fluid, earwax, endolymph, perilymph, gastric juice, mucus, ascites, pleural fluid, saliva, sebum (skin oil), semen, sweat, tears, vaginal secretions, or vomit samples containing components or fractions thereof. The bodily fluid samples may be mixed or pooled. Thus, the bodily fluid sample may be a mixture of a blood sample and a urine sample, or a mixture of a blood sample and a cerebrospinal fluid sample. The sample may also be homogenized by filtration, dilution and / or centrifugation, etc. In some embodiments, the sample is skin, hair, cells, or tissue.

[0140] item The present invention may be further defined by the following items: 1. A method for determining the methylation state of at least one target nucleotide (NOI) in a target nucleic acid sequence, wherein the target nucleic acid sequence includes a target anchor sequence containing the NOI, and this method is a. A step of providing an oligonucleotide comprising an anchor sequence (An), wherein the anchor sequence is at least 50% complementary to a target anchor sequence, and the anchor sequence comprises at least one hydrophobic nucleotide (H) located between a nucleotide complementary to the NOI and a nucleotide in the oligonucleotide immediately 5' to the NOI; b. If the NOI is not methylated, the step is to incubate the oligonucleotide with the target nucleic acid at a temperature higher than the melting temperature between the oligonucleotide and the target nucleic acid sequence of interest. c. A step of detecting whether the oligonucleotide anneals to the target nucleic acid of the desired value, This includes the step of determining the methylation state, Here, the hydrophobic nucleotide (H) has the following structure; XYQ (In the formula, X is a skeletal monomer unit that can be incorporated into the backbone of a nucleotide or nucleotide analog, or a nucleic acid or nucleic acid analog. Q is an intercalator that does not participate in Watson-Crick hydrogen bonding; Y is a linker portion that connects a nucleotide or nucleotide analog or backbone monomer unit to an intercalator. method.

[0141] 2. The method according to item 1, wherein the target nucleic acid is DNA.

[0142] 3. A method according to any one of the preceding items, wherein the NOI is cytosine.

[0143] 4. The method according to any one of items 3-4, wherein cytosine is located at the CpG site and the anchor sequence contains the sequence-CHG-.

[0144] 5. The method according to any one of the preceding items, wherein the anchor sequence comprises at least two, e.g., at least three, e.g., at least four, e.g., in the range of 2 to 10, e.g., in the range of 2 to 5, e.g., in the range of 3 to 5, e.g., in the range of 4 to 5 -NHG- sequences, where each N is individually selected from the group consisting of C, G, A, and T, and each sequence -NG- is complementary to the cytosine of interest and its adjacent nucleotides.

[0145] 6. The method according to any one of the preceding items, wherein steps b) and c) together include performing PCR, and the temperature is used as the annealing temperature for one or more cycles of PCR.

[0146] 7. The oligonucleotide has the structure 5'-An-Lp-St-3', in the formula, An is an anchor array; Lp is a loop sequence and is not complementary to the target nucleic acid sequence. St is a starter sequence, which is at least 90% complementary to the target starter sequence, and the target starter sequence is the target nucleic acid sequence located at 5' of the target anchor sequence. A method described in any one of the preceding items.

[0147] 8. The method according to any one of the preceding items, wherein the anchor sequence (An) consists of nucleotides in the range of 8 to 30, for example, 8 to 20, for example, 10 to 20, for example, 15 to 20, and one or more of the nucleotides are hydrophobic nucleotides.

[0148] 9. The method according to any one of the preceding items, wherein the anchor sequence comprises sequence-NHG-, where N is selected from the group consisting of C, G, A, and T, and sequence-NG- is complementary to the cytosine of interest and its adjacent nucleotides.

[0149] 10. The method according to any one of items 8 to 9, wherein Lp and St together consist of at least 15 nucleotides, for example, at least 17 nucleotides, for example, in the range of 15 to 45 nucleotides, for example, in the range of 17 to 35 nucleotides, for example, in the range of 17 to 25 nucleotides.

[0150] 11. A method according to any of the preceding items, wherein the target nucleic acid sequence is present in DNA purified from a sample.

[0151] 12. A method for determining the risk of an individual having a clinical condition, or the risk of an individual developing a clinical condition, wherein the clinical condition is related to the methylation status of NOI in the nucleic acid of interest, and this method is a. Providing samples from individuals, b. Determine the methylation status of NOI in the sample by performing one of the methods described in the preceding items, Methods that include...

[0152] 13. A method for determining the potential effect of treating a clinical condition in an individual requiring it, wherein the effect of treating the clinical condition is related to the methylation state of NOI in the nucleic acid of interest, and this method is a. Providing samples from individuals, b. Determine the methylation status of NOI in the sample by performing one of the methods described in items 1 to 10, Methods that include...

[0153] 14. The clinical condition is cancer, and the method described in any one of items 12-13.

[0154] 15. Oligonucleotides containing or consisting of the following general structures: 5'(N) n -HG-(N) m -HG-(N) p -3' (wherein N is any nucleotide or nucleotide analogue; G is the nucleotide guanine; n is a non-negative integer; m is an integer greater than or equal to 1; p is a non-negative integer; H is a hydrophobic nucleotide, Here, the hydrophobic nucleotide has the following structure; XYQ During the ceremony, X is a skeletal monomer unit that can be incorporated into the backbone of a nucleotide or nucleotide analog, or a nucleic acid or nucleic acid analog. Q is an intercalator that does not participate in Watson-Crick hydrogen bonding; Y is a linker portion that connects a nucleotide or nucleotide analog or backbone monomer unit to an intercalator.

[0155] 16. The method or oligonucleotide according to any one of the prior claims, wherein at least one Q, such as all intercalators, is selected from the group consisting of polyaromates and heteropolyaromates optionally substituted with one or more selected from the group consisting of hydroxyl, bromo, fluoro, chloro, iodo, mercapto, thio, cyano, alkylthio, heterocyclic, aryl, heteroaryl, carboxyl, carboxyl, alkyl, alkenyl, alkynyl, nitro, amino, alkoxyl, and amide.

[0156] Examples The present invention will be further illustrated by the following embodiments, but these should not be construed as limiting the invention.

[0157] Example 1 MGMT promoter area The desired sequence for the MGMT promoter region is included herein as Sequence ID No. 1: [ka]

[0158] The sequence is 129467120-129467385, human chromosome 10, GRCh38.p12. The four underlined CpG sites in Sequence ID No. 1 are the CpG sites for which this assay is designed to analyze their methylation status. Three of the four selected CpG sites are commonly used for methylation detection in commercially available kits, such as Qiagen's pyrosequencing kit (Qiagen. (2014) EpiTect (registered trademark) Fast Bisulfite Conversion Handbook For sample lysis and complete bisulfite Sample&Assay Technologies QIAGEN Sample and Assay Technologies. Sample&Assay Technologies).

[0159] Common PCR assay settings The following design was used for all PCR assay setups except for Examples 12 and 13. The volume is for one PCR tube containing a total reaction volume of 25 μL. 1) 12.5 μL of 2x master mix* (MM) containing DNA polymerase, dNTPs, MgCl2, and buffer. 2) 7.5 μL of primer / probe (PP) mixture 3) 5 μL of purified genomic DNA

[0160] In Examples 12 and 13, the following design was used to set up the PCR assay. The volume is for one PCR tube containing a total reaction volume of 12 μL. 1) 6 μL of 2x Master Mix* (MM) containing DNA polymerase, dNTPs, MgCl2, and buffer. 2) 1 μL of primer / probe (PP) mixture 3) 5 μL of purified genomic DNA *Unless otherwise specified, the master mix (MM) consists of 2x Ampliqueen (available from PentaBase ApS). The concentration of MgCl2 is 2.25 mM in the Ampliqueen working solution.

[0161] All PCRs are performed using one of the following real-time PCR instruments: MyGoPro (It-IsLifeScienceltd), BaseCycler, or BaseTyper (PentaBase ApS). RFU is determined using the standard settings of the PCR instrument.

[0162] T m and ΔT m The melting temperature of an oligonucleotide is determined experimentally by incubating the oligonucleotide with its complementary target while increasing the temperature. Generally, oligonucleotides are labeled with a fluorescent label so that the fluorescent signal is binding-dependent. For example, an oligonucleotide may be bound to a fluorescent moiety at one end and to a quencher at the other end. At low temperatures, the probe binds to the target and forms a double helix, thereby generating a fluorescent signal, which is measured as RFU. As the temperature increases, the RFU decreases, and the T of the double helix decreases. m When this point is reached, a sharp decrease in RFU may be detected. This is shown in Figure 1A. m Then, half of the nucleotides bound at the start of the melting process dissociate from the target. The first negative derivative of the melting curve can be plotted, and the peak (maximum) of this curve is the melting temperature of the probe (Figure 1B).

[0163] T m The difference is calculated as the difference obtained by subtracting the value of the same probe bound to one target from the value of the same probe bound to another target.

number

[0164] Calculation of ΔCt and ΔΔCt Ct refers to the threshold cycle, i.e., the PCR cycle in which a product is detected based on a predetermined criterion. Generally, Ct is determined based on the fluorescence threshold. Unless otherwise specified, the default settings of the PCR machine MyGoPro, BaseCycler, or BaseTyper and their associated software are used. The threshold is set automatically by the software.

[0165] ΔCt is calculated between a reference assay (i.e., an assay using a reference primer) and a methylation-sensitive assay (i.e., an assay using a methylation-specific base primer containing a hydrophobic nucleotide).

number

[0166] ΔΔCt is calculated as the difference between the ΔCt of the assay with unmethylated DNA added and the ΔCt of the assay with methylated DNA added as a template.

number

[0167] Therefore, ΔCt non.mC This shows the difference in Ct between the reference assay and the test assay when using a non-methylated DNA template. ΔCt non.mC In this specification, this is also referred to as Δnon.mC. ΔCt non.mC This indicates the specificity of the assay, and it is preferable that it be as high as possible.

[0168] ΔCt mC This shows the difference in Ct between the reference assay and the methylation-specific assay using a methylated DNA template. In this specification, ΔCt mC This is also called ΔmC. ΔCt mC This indicates the assay sensitivity, which is preferably as low as possible.

[0169] ΔΔCt is ΔCt non.mC and ΔCt mC This is the difference between the two, and this should preferably be as high as possible.

[0170] Explanation of base primer The base primer approach is based on the selective amplification of methylated DNA using hydrophobic nucleotides. This selection can be achieved by a single primer, which is referred to herein as the base primer. The base primer consists of three parts: an anchor sequence, a loop sequence, and a starter sequence. The anchor sequence contains hydrophobic nucleotides. The anchor sequence has high thermal stability when bound to methylated DNA compared to unmethylated DNA. Therefore, the anchor sequence is used to provide stability for the starter sequence, which is located at the 3' end of the primer. The starter sequence should have low thermal stability and therefore will not bind to the DNA template if it does not receive support from the anchor sequence (Figure 4B). PCR is initiated only when the anchor sequence binds with high thermal stability, which allows the starter sequence to bind (Figure 3A, "Cycle 1"). It should be noted that polymerase generally adds dNTPs containing unmethylated nucleotides in the PCR reaction, so the amplicon created during PCR will not contain any methylated nucleotides.

[0171] The anchor sequence and starter sequence are connected by a loop sequence. This sequence is beneficial because most polymerases cannot read hydrophobic nucleotides, and therefore the anchor sequence is cleaved when the reverse primer binds and a complementary chain is created. The starter sequence is designed to have low thermal stability and therefore cannot bind to the amplicon without the additional sequence. Thus, the loop sequence is incorporated into the primer, so that after two cycles of methyl-specific amplification, the loop sequence and starter sequence can function as a normal primer (Figure 3A, "Remaining Cycles").

[0172] Example 2 Affinity for 5-methylcytosine Oligonucleotides complementary to the region containing the four CpGs marked in Sequence ID No. 1 above were synthesized. The probes were double-labeled with a FAM fluorophore at the 5' end and a black hole quencher (BHQ) at the 3' end. The probes used two different hydrophobic nucleotides, either Z-type or E-type.

[0173] The Z-type hydrophobic nucleotide has a phosphoramidite with the chemical name 3-(1-O-(4,4'-dimethoxytriphenylmethyl)-2-O-(2-cyanoethyldiisopropylamidephosphite)-1,2-butanediol)-4-N-(7,9-dimethyl-3H-pyrido[3',2':4,5]thieno[3,2-d]pyrimidine-4-one). In the Z-type hydrophobic nucleotide, the intercalator Q has the following structure:

[0174] [ka]

[0175] In this specification, a hydrophobic nucleotide of type Z may be simply referred to as "Z".

[0176] The hydrophobic nucleotide of type E has a phosphoramidite with the chemical name (S)-1-(4,4'-dimethoxytriphenylmethyloxy)-3-pyrenemethyloxy-2-propanol. In this hydrophobic nucleotide of type E, the intercalator Q has the following structure:

[0177] [ka]

[0178] In this specification, hydrophobic nucleotides of type E may be simply referred to as "E".

[0179] Either Z or E was incorporated into the probe in such a way that it was positioned between C and G in the four CpGs of Sequence ID No. 1. This was done to test which of E and Z best distinguished between methylated DNA (referred to as "5-mC") and unmethylated DNA (referred to as "non.mC").

[0180] Furthermore, E was tested at various positions. Probes containing E located between C and G in the CpG are referred to herein as E-type probes. Probes in which E is located immediately before the CpG during annealing are referred to as E2-type probes, and probes in which E is located immediately after the CpG during annealing are referred to as E3-type probes. For reference, probes were also fabricated without E and Z. Table 1 shows the arrangements of the different probes.

[0181] Two artificial targets were designed and synthesized. One does not contain any methylated cytosine (mC), and the other contains mC at four CpG sites. The sequences are shown in Table 1. [Table 1]

[0182] The melting temperature was determined as described in Example 1.

[0183] Probes containing hydrophobic nucleotides E and Z exhibit high T when bound to fully methylated targets (MGMT_1,2,3,4mC) compared to unmethylated targets (MGMT_0mC). m It was observed that it possessed the following characteristics. The difference in melting temperature between 1,2,3,4mC and 0mC was approximately 6.6°C for probe MGMT_Z and approximately 9.6°C for probe MGMT_E. Furthermore, when the reference probe (MGMT_Ref) was hybridized to a methylated target, the difference between the mC target and the non-mC target was 3.4°C, indicating high T m It was observed that the following characteristics were present. See Figure 4 and Table 2.

[0184] The position of the hydrophobic nucleotide E type was examined. The best discrimination was achieved when E was placed between C and G of CpG (probe MGMT_E). When the hydrophobic nucleotide was placed after CpG (probe MGMT_E3), less discrimination was observed than in the case of the reference probe.

[0185] The differences in the first negative derivatives of the melting of MGMT_E and MGMT_Ref with methylated targets (MGMT_1,2,3,4mC) and unmethylated targets (MGMT_0mC) are shown in Figure 5. [Table 2]

[0186] Example 3 Affinity of Z and E modified probes related to the position and number of 5-mC The correlation between the melting temperatures of Z and E modified probes with complementary targets and the position and number of 5-mC in the target oligo was investigated. Four different targets each containing one methylated CpG site were designed.

[0187] Furthermore, one target having two methylated CpG sites was designed and another target having four methylated CpG sites was designed. This was done to investigate the correlation between the number of 5mC and the melting temperature of the probe. The sequences are shown in Table 3. [Table 3]

[0188] No significant difference was observed between the positions of the methylated sites in the target at the melting temperature.

[0189] The correlation between the number of methylated sites of the probe and the melting temperature was investigated. The R of the linear fit when unmethylated targets were excluded 2 was R of 0.997 2The following was obtained. This indicates a linear correlation when the target is methylated. The unmethylated target had a lower melting temperature to fit the linear model. The plot is shown in Figure 6, and the raw data is shown in Table 4. [Table 4]

[0190] Example 4 Melting test with Ampliqueen Master Mix Melting the double-stranded MGMT_E / 1,2,3,4mC double-stranded MGMT_E / 0 [Table 5]

[0191] Example 5 Base primer design Four different base primer designs were prepared (Table 6). The primers were designed to have two different loop sequences and two different lengths of 3'-end starter sequences. The melting temperatures were determined by the T of the loop sequences and 3'-end starter sequences. m This is calculated based on the fact that after the loop sequences are incorporated into the amplicon, these sequences function as a single primer. [Table 6] [Table 7]

[0192] Base Primer Test PCR was set up using the base primers in Table 6 as forward primers and the reverse primer MGMT_Rev2C (Table 4). In the reference assay, primer MGMT_Fw2C was used as the forward primer and MGMT_Rev2C as the reverse primer (Table 7).

[0193] The methylation-specific assay was performed by mixing 700 nM of each base primer in Table 7 with 700 nM MGMT_Rev2C and 500 nM MGMT_Probe2E (Table 7). The reference assay was conducted by mixing 700 nM MGMT_Fw2C with 700 nM MGMT_Rev2C and 500 nM MGMT_Probe2E. The PCR program is shown in Table 8, and the optimization protocol for optimizing the PCR program is shown in Table 9.

[0194] Only MGMT_FwLoop3C and FwLoop4C were used for optimizing the program, and only FwLoop4C was tested at 79°C. In this experiment, a master mix containing 1% ZUBR Green was used for signal generation. 1 ng / μL unmethylated DNA and 1 ng / μL methylated DNA (purified from the cell line IDH U87 WT) from healthy donors were used in the experiment. [Table 8] [Table 9]

[0195] All base primers were found to be useful for DNA amplification. MGMT_FwLoop3B and FwLoop4B were found to have high Ct values ​​in the methyl-specific assay compared to the reference assay, meaning that the base primers amplified methylated DNA with low efficiency. MGMT_FwLoop3C and FwLoop4C had low ΔCt in methylated DNA, meaning that the base primers amplified methylated DNA with nearly the same efficiency as the reference assay. However, FwLoop3C and FwLoop4C did not distinguish well between methylated and unmethylated DNA (ΔΔCt < 0.5). The two base primers were tested with shorter annealing times and higher temperatures, and the distinction improved (see Figure 8).

[0196] At both annealing temperatures of 77°C and 79°C, a decrease in ΔCt for both methylated and unmethylated DNA was observed when the annealing time was extended from 20 seconds to 30 seconds (Figure 8). This indicates that the base primer requires more time to anneal efficiently to the methylated template. The highest ΔΔCt was 4.46 ± 0.86 at 79°C for 30 seconds.

[0197] Example 6 Methyl-specific pre-amplification MGMT_FwLoop4C was tested using a PCR program with methyl-specific pre-amplification (Table 10). The methyl-specific assay was performed by mixing 700 nM MGMT_FwLoop4C with 700 nM MGMT_Rev2C. The reference assay was performed by mixing 700 nM MGMT_Fw2C with 700 nM Rev2C. A master mix containing 1% ZUBR Green was used for both the reference assay and the methyl-specific assay. The results were compared to a standard two-step amplification with annealing settings of 20 seconds at 79°C. The same experimental settings as in the above examples were used. [Table 10]

[0198] ΔCt and ΔΔCt were determined as described in Example 1 above.

[0199] Methyl-specific pre-amplification cycling lowered the Ct value of methylated DNA and increased the Ct value of unmethylated DNA in the methyl-specific assay, thereby improving methylation discrimination to a ΔΔCt value of 9.93. See Table 11. The PCR curve for methyl-specific pre-amplification PCR is shown in Figure 9. However, the Ct value of methylated DNA in the methyl-specific assay remained high in pre-amplification, suggesting that the loop sequence and starter sequence were not sufficiently amplified in the two-step unmethyl-specific amplification. [Table 11]

[0200] Example 7 higher T m Design of base primer The base primers were designed with loop sequences that have higher affinity to the complementary sequence and the 3' end starter sequence, allowing them to work at high temperatures in two-step amplification. The designs are shown in Table 12. Instead of using conventional primers, the affinity to MGMT_Fw2C and MGMT_Rev2C was increased by adding a hydrophobic nucleotide to the 5' end of these primers. [Table 12]

[0201] The new base primers were initially analyzed without pre-amplification to find the optimal annealing time and temperature in two-step amplification. Instead, the assay was mixed using MGMT_FwLoop5C as the forward primer, as described in "Methyl-Specific Pre-Amplification". PCR program D was modified according to Table 13. The SW1088 cell line was used in this experiment and subsequent experiments. [Table 13]

[0202] ΔCt and ΔΔCt were determined as described in Example 1 above.

[0203] Figure 10 shows the results obtained for ΔCt and ΔΔCt at different annealing times and temperatures for the base primer MGMT_FwLoop5C. It was found that increasing the annealing temperature (fixed at 30 seconds) worsened methyl-specific amplification of both methylated and unmethylated DNA. The best differentiation between methylated and unmethylated DNA was observed at 80°C.

[0204] Example 8 Titration of MGMT_FwLoop5C The base primers were tested in methyl-specific pre-amplification using PCR program E, with 81°C used in the second stage of pre-amplification and both 79°C and 80°C used in the second stage of two-stage amplification. [Table 14]

[0205] Using PCR program E, MGMT_FwLoop5C was titrated at concentrations of 500 nM, 700 nM, and 900 nM, with 81°C used for the second stage of pre-amplification and 80°C for the second stage of two-stage amplification. Except for the concentration variations, the assay was performed as described in section "Higher T m The design of the base primer was carried out as described in "Design of the base primer".

[0206] MGMT_FwLoop5C was analyzed using methyl-specific pre-amplification. 500 nM FwLoop5C was the optimal base primer concentration. Here, ΔΔCt was 12.6, and the ΔCt of methylated DNA was 9.8. Higher concentrations resulted in worse methyl-specific amplification. See Table 14 for results. [Table 15]

[0207] Example 9 1. Test for specific amplification of retained methyl groups. Instead of methyl-specific pre-amplification, MGMT_FwLoop5C was used to investigate one-step methyl-specific retention. The assay used 500nM FwLoop5C instead of 700nM in section "Higher T m The base primer design was carried out as described in "Design of Base Primers". PCR program F was used (Table 15). Methyl retention times were tested at 60 seconds and 120 seconds at 80°C. [Table 16]

[0208] To improve the specificity of the base primer, the 5-cycle methyl-specific amplification was changed to methyl retention. At 80°C for 60 seconds (see Figure 11), this proved superior to the 5-cycle methyl-specific amplification shown in Table 14. ΔΔCt increased from 12.6 to 14.8. Raw data are shown in Table 16. [Table 17]

[0209] Example 10 Verification of the method The method was further validated by evaluation of patient samples using linearity and sensitivity tests. All validation samples were double-tested with PCR program F. The methyl-specific assay was performed by mixing 500 nM MGMT_FwLoop5C with 700 nM MGMT_Rev2C_suE. The reference assay was performed by mixing 700 nM MGMT_Fw2C_suE with 700 nM Rev2C_suE. A master mix containing 1% ZUBR Green was used for both the reference assay and the methyl-specific assay.

[0210] Linearity of reference and methyl-specific assays Linearity tests were performed on methylated templates (DNA purified from SW1088 cells) and unmethylated templates (DNA purified from the blood of healthy patients). The templates were diluted to concentrations of 50, 25, 12.5, 6.25, 3.13, 1.56, and 0.78 ng / μL and added to both the reference and methyl-specific assays. The standard curve was fitted to the following equation (Kubista et al., 2006):

number

number

[0211] Linearity was superior in the reference assay compared to the methyl-specific assay. Linearity was superior in the methylation template (R) on the reference assay. 2 Compared to the non-methylated template (R = 0.98), 2 The non-methylated template (R = 0.998) was superior. The opposite result was observed in the methyl-specific assay, and linearity was better with the non-methylated template (R 2 Compared to the methylation template (R = 0.811), 2 The best result was (=0.873). See Figure 12.

[0212] PCR efficiency was over 100% with the non-methylated template and less than 100% with the methylated template. This explains both the reference assay and the methyl-specific assay (FwLoop5C). The results are shown in Table 17. [Table 18]

[0213] Sensitivity of methyl-specific assays The sensitivity of the methyl-specific assay was analyzed by mixing 2 ng / μL of purified DNA from SW1088 with 2 ng / μL of purified DNA from the blood of healthy patients. The following concentrations were prepared: 100%, 50%, 25%, 10%, 5%, 1%, and 0% methylation. Sensitivity was performed using a MyGo Pro instrument.

[0214] The sensitivity was 5%, and clear differentiation between methylated and unmethylated DNA could be established at ΔCt = 18. No linearity was observed between the methylation rate (%) and ΔCt (see Figure 13).

[0215] Based on the results, we can propose a rough classification of patients into hypermethylated, hypomethylated, and unmethylated categories based on ΔCt. See Table 18. [Table 19]

[0216] Example 11 Melting test of mutation (mismatch) probes Melt-breading tests were performed using the MGMT_E probe by intentionally adding several mismatched nucleotides to the sequence. Two probes were constructed with two mutations (leading to two mismatches), and three probes were constructed with one mutation (leading to one mismatch). The sequences are shown in Table 20. The experimental setup was the same as in the section "Affinity for 5-methylcytosine". [Table 20]

[0217] Probes with a mismatch with the target sequence, with the exception of MGMT_E_M2, exhibit greater specificity (ΔT) towards methylated DNA than towards unmethylated DNA. m It was found that this lowers the melting temperature of the double-stranded structure without affecting ΔT, and here, ΔT mA slight decrease was observed. The results are shown in Figure 14, and the raw data are shown in Table 21. This experiment demonstrates that it is possible to reduce the affinity of methylation-specific primers spanning several high-affinity CpG sites, for example, without reducing specificity. [Table 21]

[0218] Example 12 Sensitivity of base primers with a single mismatch in the anchor sequence Base primers with a single mismatch in the anchor sequence were evaluated using sensitivity tests performed on synthetic DNA from the MGMT promoter region. Synthetic DNA was methylated using CpG methyltransferase (New England Biolabs Inc, Ipswich, MA, USA) according to the manufacturer's protocol. Fully methylated and unmethylated DNA were mixed at dilution concentrations of 500 copies / μL to obtain methylation templates of 100%, 50%, 25%, 12.5%, 6.3%, 3.1%, and 0%. 700nM MGMT_Fw2C, 700nM MGMT_Rev2E, and 500nM MGMT_Probe2E were used in the reference assay, while 700nM MGMT_FwLoop5D_M5 was used instead of MGMT_Fw2C for the methyl-specific assay (see Table 22 for sequences). This test was performed using PCR program G (see Table 23) on BaseTyper® real-time PCR instruments (PentaBase, Odense, Denmark). [Table 22] [Table 23]

[0219] The lowest detectable sample contained 6.3% methylated DNA, and a linear correlation was observed between ΔCt and log(methylation rate%) (see Figure 15). Based on this linear correlation, it is possible to quantify the degree of methylation based on the ΔCt value.

[0220] Investigation of the number of base pairs between anchor sequences and starter sequences In the aforementioned base primers, the starter sequence was complementary to the target sequence immediately at the 3' end of the anchor sequence. A novel base primer with a single mismatch in the anchor sequence was designed to have a nucleotide gap between the 3' end of the anchor sequence on the complementary target sequence and the 5' end of the starter sequence (see Figure 16). The base primers were designed using two starter sequences of different lengths (A and B) to have gaps of 0, 2, 4, 6, and 8 nucleotides. See Table 24. [Table 24]

[0221] 700nM MGMT_Fw2C, 700n MMGMT_Rev2E, and 500nM MGMT_Probe2E were used in the reference assay, and for methyl-specific assays, the 700nM base primers listed in Table 24 were used instead of MGMT_Fw2C. 500 copies / μL of unmethylated, 100% methylated, and 10% methylated synthetic DNA were used as templates. PCR was performed on a BaseTyper using PCR program H (see Table 25). [Table 25]

[0222] With the exception of MGMT_FwLoop5B_M5_8A and MGMT_FwLoop5B_M5_8B, all designs were found to be able to distinguish between 100% mC, 10% mC, and non-mC DNA (see Figure 17).

[0223] Example 13 Auxiliary primer complex

[0224] We tested an alternative methyl-recognition primer design. The methyl-recognition primer complex consists of an auxiliary primer containing an anchor sequence and a stem sequence. The rest of the complex is a standard primer, partially complementary to the stem sequence of the auxiliary primer and partially complementary to the target sequence. Recognition of methylated DNA from unmethylated DNA is performed by the same principle as with the base primer. However, after PCR is initiated with methylated DNA, the standard primer is used for the remainder of the cycle. This principle is illustrated in Figure 18.

[0225] The auxiliary primer complexes were tested with the sequences shown in Table 26. For the reference assay, 700nM MGMT_Fw2C, 700nM MGMT_Rev2E, and 500nM MGMT_Probe2E were used. For the methyl-specific assay, 700nM MGMT_Primer_1D and 50nM MGMT_Assist_1A_E were used instead of MGMT_Fw2C. 500 copies / μL of unmethylated and 100% methylated synthetic DNA were used as templates. PCR was performed on a BaseTyper using PCR program H (see Table 25). [Table 26]

[0226] The PCR curves are shown in Figure 19. ΔCt was 5.6 for 100% methylated DNA and 9.5 for unmethylated DNA, and ΔΔCt was 3.9.

Claims

1. A method for determining the methylation state of at least one target nucleotide (NOI) in a target unmodified target nucleic acid sequence, wherein the target nucleic acid sequence includes a target anchor sequence containing the NOI, and the method is a. A step of providing an oligonucleotide comprising an anchor sequence (An), wherein the anchor sequence is at least 50% complementary to the target anchor sequence, and the anchor sequence comprises at least one hydrophobic nucleotide (H) located between the nucleotide complementary to the NOI and the nucleotide immediately 5' to it; b. The step of incubating the oligonucleotide with the target nucleic acid at a temperature higher than the melting temperature between the oligonucleotide and the target nucleic acid sequence when the NOI is not methylated, c. A step of detecting whether the oligonucleotide anneals to the target nucleic acid of the objective, This includes the step of determining the methylation state, i. Here, the hydrophobic nucleotide (H) has the following structure X-Y-Q It has, in the formula, X is a skeletal monomer unit that can be incorporated into the backbone of a nucleotide or nucleotide analog, or a nucleic acid or nucleic acid analog, wherein the skeletal monomer unit includes a phosphoramidite; Q is an intercalator that does not participate in Watson-Crick hydrogen bonding; Y is a linker portion that connects a nucleotide or nucleotide analog or backbone monomer unit to the intercalator. ii. The oligonucleotide has the structure 5'-An-Lp-St-3', In the formula, An is the anchor array; Lp is a loop sequence that is not complementary to the target nucleic acid sequence, and the loop sequence consists of a single nucleic acid sequence capable of forming a protruding structure, or consists of two or more nucleic acid sequences that can at least partially hybridize with each other to form a complex capable of forming a protruding structure; A method wherein St is a starter sequence that can be hybridized to a target starter sequence, the target starter sequence being the sequence of the target nucleic acid sequence located at 5' of the target anchor sequence.

2. The method according to claim 1, wherein the target nucleic acid for the purpose is DNA or RNA.

3. The method according to any one of claims 1 to 2, wherein, prior to carrying out the method, the target unmodified nucleic acid sequence has not been subjected to a process including bisulfite conversion, restriction enzyme digestion, or TET enzyme conversion.

4. The method according to any one of claims 1 to 3, wherein the NOI is cytosine.

5. A. i. An oligonucleotide comprising the hydrophobic nucleotide(s) when hybridized to the target nucleic acid sequence, wherein the target nucleic acid sequence contains at least one methylated NOI; ii. The absolute difference in melting temperatures between the oligonucleotide containing the hydrophobic nucleotide(s) when hybridized to the target nucleic acid sequence, wherein the target nucleic acid sequence does not contain methylated NOI, and B. i. An oligonucleotide identical to the oligonucleotide in A., but which, when hybridized to the target nucleic acid sequence, does not contain the hydrophobic nucleotide(s), and the target nucleic acid sequence contains the same number of methylated NOIs as A. i.; and ii. An oligonucleotide identical to the oligonucleotide in A., but which, when hybridized to the target nucleic acid sequence, does not contain the hydrophobic nucleotide(s), and the target nucleic acid sequence does not contain methylated NOI; the absolute difference in melting temperature between this oligonucleotide and the target oligonucleotide is: The method according to any one of claims 1 to 4, wherein the difference is at least 0.50°C per NOI that is methylated instead of unmethylated in the target nucleic acid sequence of the objective, where the difference is A - B, the difference is a positive difference, and the melting temperature is measured in a TM buffer containing 0.02 M Na2HPO4, 0.02 M NaCl, and 2 mM EDTA.

6. The method according to claim 4, or the method according to claim 5, referencing claim 4, wherein the anchor sequence comprises at least one -N-H-G- sequence, where each -G- is complementary to the cytosine of interest, each -N- is individually selected from the group consisting of C, G, A, and T and is complementary to the adjacent nucleotide of the cytosine of interest, and H is a hydrophobic nucleotide.

7. The method according to any one of claims 1 to 6, wherein step b) and c) both include performing PCR, and the temperature is used as the annealing temperature for one or more cycles of the PCR.

8. The method according to any one of claims 1 to 7, wherein the oligonucleotide comprises a first and a second nucleic acid sequence, the first nucleic acid sequence comprising an anchor sequence (An) complementary to the target nucleic acid sequence and a first portion of a loop sequence not complementary to the target nucleic acid sequence, the second nucleic acid sequence comprising a second portion of the loop sequence that can at least partially hybridize to the first portion of the loop sequence, the second nucleic acid sequence further comprising a starter sequence (St), and the first and second nucleic acids, when hybridized, can form a protruding structure.

9. The method according to any one of claims 1 to 8, wherein the target anchor sequence and the target starter sequence are located within 10 nucleotides of each other.

10. The method according to any one of claims 1 to 9, wherein Lp and St are selected such that the oligonucleotide consisting of Lp-St has a melting temperature of at least 50°C together with its complementary sequence.

11. The method according to any one of claims 1 to 10, wherein the difference in melting temperature between the oligonucleotide and the target methylation target nucleic acid sequence is at least 5°C higher than the difference in melting temperature between the oligonucleotide and the target unmethylation target nucleic acid sequence.

12. The method according to any one of claims 1 to 11, wherein the difference in melting temperature between the oligonucleotide and the methylated target nucleic acid sequence, compared to the target unmethylated target nucleic acid sequence, is at least 1°C higher than the difference in melting temperature between an oligonucleotide with the same sequence as the unmethylated target nucleic acid sequence, except that it lacks the hydrophobic nucleotides, and the methylated target nucleic acid sequence.

13. PCR comprises one or more methyl-specific amplification cycles and one or more general amplification cycles, wherein the methyl-specific amplification cycle(s) are a. The step of dissolving nucleic acids, b. A step comprising annealing and extension under methyl-specific conditions, The aforementioned general amplification cycle, a. The step of dissolving nucleic acids, b. Including annealing and extension steps under general conditions, The method according to any one of claims 7 to 12, wherein the melting comprises incubation at a temperature of at least 90°C, and the annealing and extension under methyl-specific conditions are carried out at a higher temperature than annealing and extension under general conditions.

14. The method according to any one of claims 7 to 13, wherein PCR is performed using the oligonucleotide as a forward primer, and the PCR further comprises using a reverse primer which is at least 90% identical to a sequence downstream of the target nucleic acid sequence of the object.

15. The method according to any one of claims 1 to 14, wherein the target nucleic acid sequence of interest is contained in DNA purified from a sample obtained from an individual suffering from or at risk of having a clinical condition related to the methylation of the target nucleic acid.

16. The method according to any one of claims 1 to 15, wherein the oligonucleotide comprises or consists of the following general structure: 5’(N) ｎ -HG-(N) ｍ -HG-(N) ｐ -HG-(N) ｑ -3’ (wherein N is a nucleotide or a nucleotide analog; G is the nucleotide guanine; n is a non-negative integer; m is an integer greater than or equal to 1; p is a non-negative integer; q is a non-negative integer; H is a hydrophobic nucleotide), or 5’(N) ｎ -CGH-(N) ｍ -CGH-(N) ｐ -3’ (wherein N is a nucleotide or a nucleotide analog; C is the nucleotide cytosine; G is the nucleotide guanine; n is a non-negative integer; m is an integer greater than or equal to 1; p is a non-negative integer; H is a hydrophobic nucleotide.

17. The method according to claim 16, wherein the oligonucleotide comprises or consists of the following general structure: 5’(N) ｎ -HG-(N) ｍ -HG-(N) ｐ -HG-(N) ｑ -HG-(N) ｕ -3’ (In the formula, p is an integer greater than or equal to 1; q is an integer greater than or equal to 1; u is a non-negative integer), or 5’(N) ｎ -CGH-(N) ｍ -CGH-(N) ｐ -CGH-(N) ｑ -CGH-(N) ｕ -3’ (In the formula, p is a non-negative integer; q is an integer greater than or equal to 1; (where u is a non-negative integer).

18. The method according to any one of claims 16 to 17, wherein the nucleotide located immediately 5' to at least one H is cytosine (C).

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