KR20220096382A - Method and kits for detecting methylation of gene using non-methylation specific probe - Google Patents

Abstract

The present invention relates to a gene methylation detection method and a detection kit using a probe specific for an unmethylated gene. The gene methylation detection method can detect gene methylation simply without treating bisulfite, and can be detected with excellent sensitivity and accuracy, and thus is useful in various disease diagnosis fields comprising cancer.

Description

Method and kits for detecting methylation of gene using non-methylation specific probe

The present invention relates to a method and a detection kit for detecting gene methylation using a non-methylated gene probe, and more particularly, to a gene methylation from analysis of a melting curve through suppression of amplification of a target gene using a probe specific for a non-methylated gene. It relates to a method and a detection kit for detecting whether

In the genomic DNA of mammalian cells, there is a 5th base in addition to A, C, G, and T, which is 5-methylcytosine (5-mC) with a methyl group attached to the 5th carbon of the cytosine ring. 5-mC always occurs only at C of a CG dinucleotide (5'-mCG-3'), and this CG is often denoted as CpG. Most of C in CpG is methylated with a methyl group attached.

Such CpG is the most common site for extragenic changes in mammalian cells, and CpG methylation inhibits the expression of repetitive nucleotide sequences in the genome, such as alu or transposon. In 5-mC of this CpG, it is naturally deamidated and the base is changed to T. Accordingly, CpG in the mammalian genome shows only a frequency of 1%, which is much lower than the frequency that should normally appear (1/4 x 1/4 = 6.25%). .

Among CpG, there are some that appear in exceptional concentration, which are called CpG islands. A CpG island is 0.2~3 kb in length, and the distribution rate of C and G bases exceeds 50%, and the distribution rate of CpG indicates a highly concentrated region of 3.75% or more.

About 45,000 CpG islands appear in the entire human genome, and are particularly concentrated in the promoter region that regulates gene expression. In fact, a CpG island appears in the promoter of housekeeping genes, which accounts for about half of human genes (Cross, S. & Bird, A.P., Curr. Opin. Gene Develop., 5:309, 1995).

On the other hand, in normal human somatic cells, the CpG islands of these housekeeping gene promoter regions are not methylated, but imprinted genes and genes on the inactivated X chromosome are methylated so that they are not expressed during development. In the process of carcinogenesis, methylation occurs on the promoter CpG island, and the expression of the corresponding gene is impaired. In particular, when methylation occurs in the promoter CpG island of tumor suppressor genes that regulate the cell cycle or apoptosis, repair DNA, participate in cell adhesion and intercellular interaction, and inhibit invasion and metastasis , which blocks the expression and function of these genes in the same way as the mutation of the coding sequence, and as a result, the development and progression of cancer is promoted. In addition, with aging, partial methylation of CpG islands appears.

As such, methylation of the promoter CpG island of the tumor suppressor gene is an important indicator of cancer because it is a representative phenomenon that occurs at the earliest stage of cancer development. It can be used in various fields such as investigation and prediction of response to anticancer therapy.

In order to maximize the accuracy of cancer diagnosis and prognosis prediction using promoter methylation, a test capable of accurately analyzing promoter CpG island methylation is required. Currently, as standard methods, bisulfite sequencing, combined bisulfite restriction analysis (COBRA), pyrosequencing, and methylation-specific polymerase chain reaction (PCR) are being actively tried. .

However, the method using bisulfite, such as these bisulfite sequencing or complex bisulfite restriction analysis, converts unmethylated cytosine to uracil by treating bisulfite, and the already methylated 5-methyl cytosine is maintained as it is. As a method to check the methylation status of individual cytosines in DNA using Therefore, a large amount of DNA is required for detection. In addition, errors such as conversion of unmethylated cytosine to uracil or conversion to thymine instead of maintaining 5-methyl cytosine as it may occur. Due to this error, the DNA methylation detection method that requires bisulfite treatment gives false-positive results, and consequently has a problem in that accuracy, specificity, sensitivity, and reproducibility are all lowered.

As a recent attempt to solve this problem, there is a method of detecting methylation of a gene by PCR clamping using a difference in binding force between a PNA (peptide nucleic acid) probe and methylated cytosine without treatment with bisulfite. There is an advanced aspect from the conventional standardized method in that it overcomes the limitation of the detection primer design in that it does not treat bisulfite and the limitation of false positives due to base conversion errors, but this is a hypothetical overexpression caused by methylation. - With respect to the detection of hyper-methylation rather than methylation (Hypo-methylation), there is a limit in that the detection sensitivity is lowered.

The specialized marker for detecting hyper-methylation is also at a level of only 10% compared to that of hypo-methylation. It is necessary to devise a highly sensitive detection method specialized for the detection of methylation.

As a result of research in order to solve the above technical problem, the present inventors have devised a detection method showing high sensitivity to hyper-methylation using a protein and nucleic acid probe specific for a non-methylation site.

It is an object of the present invention to provide a method for detecting gene methylation with high sensitivity specialized for hyper-methylation.

In addition, there is provided a method for conveniently detecting gene methylation by performing PCR using a nucleic acid probe without bisulfite treatment.

Another object of the present invention is to provide a kit for detecting methylation of a gene using a nucleic acid probe that specifically binds to an unmethylated site.

In order to achieve the above object, the present invention comprises the steps of: treating a probe capable of specifically binding to an unmethylated site to a target gene in which methylation can occur;

treating the target gene with a methyl-affinity molecule; performing PCR (Polymerase Chain Reaction) on the target gene; and analyzing the melting curve by the PCR, calculating at least one of ΔT _m (melting temperature) and ΔC _t (cycle threshold) do.

ΔT _m = T _{m (target gene)} - T _{m (unmethylated gene consisting of the same nucleotide sequence as the target gene)} , and ΔC _t = C _{t (target gene)} - C _{t (with the same nucleotide sequence as the} target gene) _{unmethylated genes)} .

In one embodiment of the present invention, before treating the target gene with the probe, the method may further include a step of selectively cleaving the unmethylated site by treatment with a restriction enzyme.

In one embodiment of the present invention, the restriction enzyme is HgaI, HhaI, Hinp1I, HpaI, SacII, AccII, Ava, BssHII, BstUI, HpaII, AatII, AciI, AcII, AfeI, AscI, Agel, BceAIBmgBI, BsaAI, BsaHI , BsiEI, BspDI, BstBI, ClaI, HaeII, KasI, NaeI, NarI, NgoMIV, NotI, NruI, Nt.BsmAI, PaeR7I, PluTI, PmlI, PvuI, RsrII, SacII, SalI, SfoI, SgrAI, SmaI, SnaBI , TspMI, ZraI, or a combination thereof.

In one embodiment of the present invention, the methyl-affinity molecule may be tetramethyl ammonium (TMA).

In one embodiment of the present invention, the probe is any one or two or more selected from the group consisting of oligonucleotides, Locked Nucleic Acid (LNA), Peptide Nucleic Acid (PNA), and mixtures thereof.

In one embodiment of the present invention, the probe may be a modified PNA probe that does not include a base at a position complementary to a methylated base of a target gene.

In one embodiment of the present invention, the methylation of the gene may include a methylated nucleotide sequence, a hydroxymethylated nucleotide sequence, a formyl methylated nucleotide sequence, a carboxyl methylated nucleotide sequence, or a combination thereof. have.

In one embodiment of the present invention, the detection method is standard PCR, real time PCR (Real Time PCR), digital PCR, isothermal PCR (Isothermal PCR), methylation specific PCR (Methylation specific PCR), real-time methylation specific PCR ( It may be any one or more selected from Real Time Methylation specific PCR), mass PCR, DNA chip, and DNA FISH (Fluorescence in situ hybridization).

In one embodiment of the present invention, before performing the PCR, adding a methyl-affinity molecule to the amplification solution; may further include.

In one embodiment of the present invention, the detection limit of methylation of the target gene may be less than 1%.

In one embodiment of the present invention, the probe may be labeled with a reporter, a quencher, an intercalating fluorescent substance, or a combination thereof.

In one embodiment of the present invention, after the PCR is performed, the step of analyzing the sequencing of the methylated target gene; may further include.

In addition, the present invention provides a kit for use in a method for detecting methylation of a gene, including a probe capable of specifically binding to an unmethylated site of a target gene in which methylation can occur, and including the above-described characteristics.

The gene methylation detection method using a non-methylation-specific probe according to the present invention can detect whether a gene is methylated with excellent sensitivity and accuracy by directly amplifying a methylated gene, and the degree of DNA methylation and methylation with a simple one-step process It provides the advantage of being able to analyze

1 is a flowchart schematically illustrating the steps of a method for detecting methylation of a gene using an unmethylated gene probe according to the present invention.
Figure 2a is a graph of the result of measuring the value of ΔT _m between unmethylated DNA and methylated DNA according to the presence or absence of a methyl-affinity molecule according to Example 1 and Comparative Example 1 of the present invention.
2B is a graph showing the result of measuring the value of ΔT _m between unmethylated DNA and methylated DNA according to the presence or absence of a methyl-affinity molecule according to Comparative Examples 2-1 and 2-2 of the present invention.
FIG. 2c is a graph showing the results of measuring ΔT _m between unmethylated DNA and methylated DNA according to the presence or absence of a methyl-affinity molecule according to Comparative Examples 3-1 and 3-2 of the present invention.
FIG. 2D is a graph showing the results of measuring ΔT _m between unmethylated DNA and methylated DNA according to the presence or absence of a methyl-affinity molecule according to Comparative Examples 4-1 and 4-2 of the present invention.
3A is a graph showing the ΔC _t values of methylated DNA and unmethylated DNA before and after treatment with a methylated gene-specific restriction enzyme (MSRE) according to Example 2 of the present invention, respectively.
3B is a graph showing the ΔC _t values of methylated DNA and unmethylated DNA before and after treatment with a methylated gene-specific restriction enzyme (MSRE) according to Comparative Example 5 of the present invention, respectively.
4 is a graph showing the result of a sensitivity test of methylation detection PCR, (a) is a non-methylation-specific probe (abasic PNA) according to the present invention, (b) is a general PNA probe, and (c) is a DNA probe. ΔC _t between methylated and unmethylated DNA was measured.

Hereinafter, a method for detecting methylation of a gene according to the present invention and a kit for use thereof will be described in detail. Unless otherwise defined, terms used in this specification should be interpreted as content commonly understood by those of ordinary skill in the art. The drawings and embodiments of the present specification are for those of ordinary skill in the art to easily understand and practice the present invention, and content that may obscure the gist of the present invention may be omitted from the drawings and embodiments, and the present invention is not limited to the drawings and embodiments is not limited to

Also, unless otherwise defined, all technical and scientific terms have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein is for the purpose of effectively describing particular embodiments only and is not intended to limit the invention.

In the following, when it is said that any one component "includes" another component, it is not construed as being limited to only the component, unless otherwise stated, and it is understood that other components may be further included. should be

The present invention comprises the steps of (S100) treating a probe capable of specifically binding with a non-methylated base to a target gene in which methylation can occur; (S200) treating the target gene with a methyl-affinity molecule; (S300) performing PCR (Polymerase Chain Reaction) on the target gene; And (S400) analyzing the melting curve by the PCR, calculating at least one of ΔT _m (melting temperature) and ΔC _t (cycle threshold); Containing, methylation detection of a gene using a non-methylated gene probe provide a way

ΔT _m = T _{m (target gene)} - T _{m (unmethylated gene consisting of the same nucleotide sequence as the target gene)} , and ΔC _t = C _{t (target gene)} - C _{t (with the same nucleotide sequence as the} target gene) _{unmethylated genes)} .

As a non-limiting example of the present invention, the (S100) step of treating a probe capable of specifically binding to an unmethylated base to a target gene in which methylation can occur, and (S200) methyl-affinity to the target gene The steps of processing the molecule may proceed concurrently.

In a preferred embodiment of the present invention, before treating the target gene with the probe, (S50) treatment with a restriction enzyme to selectively cut the unmethylated base; may further include.

The restriction enzyme processed to the target gene is a methylation sensitive restriction enzyme (MSRE), preferably MSRE that selectively cuts unmethylated bases. Specifically, for example, HgaI, HhaI, Hinp1I, HpaI, SacII, AccII, Ava, BssHII, BstUI, HpaII, AatII, AciI, AcII, AfeI, AscI, Agel, BceAIBmgBI, BsaAI, BsaHI, BsiEI, BspDI, BstBI ClaI, HaeII, KasI, NaeI, NarI, NgoMIV, NotI, NruI, Nt.BsmAI, PaeR7I, PluTI, PmlI, PvuI, RsrII, SacII, SalI, SfoI, SgrAI, SmaI, SnaBI, SrfI, TspMI, ZraI or their It may be a combination of two or more, as long as it is a restriction enzyme that selectively cuts unmethylated bases, those known in the art may be used without limitation.

Amplification of unmethylated bases can be suppressed by treating the target gene with a restriction enzyme that selectively cuts bases of unmethylated genes before treating the target gene with a non-methylation-specific probe. Since it binds strongly to the enemy probe, amplification is suppressed according to the PNA clamping principle. As a result, it is effective for inducing amplification of the methylated gene to detect whether or not the target gene is methylated or the degree of methylation with high accuracy.

In this case, the non-methylation-specific probe may be any one or two or more selected from the group consisting of oligonucleotides, locked nucleic acid (LNA), peptide nucleic acid (PNA), and mixtures thereof. Preferably, the modified PNA probe may be a modified PNA probe that does not include a base at a position complementary to the methylated base of the target gene. As a non-limiting example of the present invention, the probe may be one in which guanine is deleted at a position where a complementary bond with cytosine, which is a base for which methylation is mainly performed, is formed.

A probe having a base deleted at the position may facilitate detection of methylation of a gene by increasing the difference in ΔT _m when PCR is performed on an unmethylated gene and a methylated gene. In particular, when the target gene is treated with a methyl-affinity molecule, the ΔT _m difference may further increase. As a specific example, the methyl-affinity molecule may be tetramethyl ammonium (TMA). The TMA more easily accesses the methylated base of the target gene and is positioned at the methylated base site, so that the target gene and probe It may weaken the bonding force between them. This may mean that the melting temperature is lowered.

The method for detecting methylation of a gene using the non-methylated gene probe of the present invention using the above characteristics is to calculate at least one of ΔT _m (melting temperature) and ΔC _t (cycle threshold) by performing the PCR and analyzing the melting curve. includes steps. In this case, it may be preferably shown that ΔT _m < 0 ℃, more preferably ΔT _m ≤ -3 ℃.

Specifically, the unmethylated gene forms a strong binding force with a probe specific thereto, thereby exhibiting a high melting temperature during the PCR. On the other hand, in the presence of a methylated base in the target gene, a strong binding force with the probe cannot be formed as above, and binding is hindered due to intra-chain access of methyl-affinity molecules, resulting in a relatively low melting temperature during PCR. . For this reason, when ΔT _m < 0 °C, it can be determined that a methylated base is present in the target gene.

In addition, as there are more methylated bases in the target gene, the ΔT _m appears larger according to the difference in binding force, so a quantitative analysis of the degree of methylation can be performed using this.

In a preferred embodiment of the present invention, methylation can be determined with high accuracy using a non-methylated standard. That is, when the ΔT _m between the target gene and the probe capable of specifically binding to the unmethylated gene is large based on the standard ΔT _m between the unmethylated standard and the probe capable of specifically binding to the unmethylated gene, It can be determined that the target gene is methylated.

In the present invention, it is possible to detect methylation with high sensitivity by treating a non-methylated gene-specific probe and a methyl-affinity molecule, and preferably, by additionally pretreating a restriction enzyme that cuts the non-methylated base site.

Hereinafter, with respect to performing a sensitivity test according to the detection method of the present invention, although it will be described in more detail in Experimental Example 1, 50%, 25%, 10%, 5%, 1%, 0.5%, 0% methylated genes and ratios As a result of a sensitivity test by mixing methylated genes, the detection limit when using the non-methylation-specific probe according to the present invention was 0.5%, confirming that the gene methylation detection method of the present invention exhibits a sensitivity of about 10 times or more compared to the conventional detection limit. could

In one embodiment of the present invention, the methylation of the gene may include a methylated nucleotide sequence, a hydroxymethylated nucleotide sequence, a formyl methylated nucleotide sequence, a carboxyl methylated nucleotide sequence, or a combination thereof. have. In this case, methylation mainly occurs at the cytosine base, and the methylated cytosine specifically means methylation at the 5th carbon of the pyrimidine ring.

The melting curve analysis according to the present invention may be performed by FMCA (Fluorescence Melting Curve Analysis) method, but is not necessarily limited thereto. The fluorescence melting curve analysis is to analyze the melting curve using a fluorescent material, and the probe specific to the target gene may be labeled with a reporter, a quencher, an intercalating fluorescent material, or a combination thereof.

The reporter is a material that emits fluorescence by absorbing and emitting light of a specific wavelength, and is a material that can be labeled with a probe to confirm whether hybridization between the target gene and the probe has been made, and the quencher absorbs the light generated by the reporter material This means a material that reduces the fluorescence intensity.

The intercalating fluorescent material is acridine homodimer and its derivatives, acridine orange and its derivatives, 7-aminoactinomycin D (7-AAD) and Derivatives thereof, Actinomycin D and derivatives thereof, ACMA (9-amino-6-chloro-2-methoxyacridine) and derivatives thereof, DAPI and derivatives thereof, dihydroethidium (Dihydroethidium) and its derivatives, ethidium bromide and its derivatives, ethidium homodimer-1 (EthD-1) and its derivatives, ethidium homodimer-2 (EthD-2) and its derivatives, ethidium Ethidium monoazide and its derivatives, Hexidium iodide and its derivatives, bisbenzimide (Hoechst 33258) and its derivatives, Hoechst 33342 and its derivatives, ho Between Hoechst 34580 and its derivatives, hydroxystilbamidine and its derivatives, LDS 751 (LDS 751) and its derivatives, propidium iodide (PI) and its derivatives, It may be any one or more selected from the group consisting of dyes (Cy-dyes) derivatives.

In one embodiment of the present invention, the detection method is standard PCR, real time PCR (Real Time PCR), digital PCR, isothermal PCR (Isothermal PCR), quantitative PCR, DNA chip, DNA FISH (Fluorescence in situ hybridization) selected from It may be any one or more.

Preferably, qPCR capable of quantitative analysis while amplifying the target gene may be used, but is not necessarily limited thereto, and the PCR may be used without limitation as long as it is used in the art.

In addition, the detection method may further include a sequencing analysis step of the methylated target gene after the PCR is performed.

The present invention also provides a kit for using the above-described method for detecting methylation of a gene, comprising a probe capable of specifically binding to an unmethylated base of a target gene in which methylation can occur.

The present invention will be described in more detail with reference to the following examples. The following examples correspond to one example for describing the present invention in more detail, and the scope of the present invention is not limited by the examples.

[Example 1]. Dissolution curve analysis of genes using Abasic PNA probes and methyl-affinity molecules

Fluorescently labeled (5'-FAM, 3'-Dabcyl) PNA probe of SEQ ID NO: 4 (Panagene, Korea) 1 μM, unmethylated DNA oligomer of SEQ ID NO: 5 having the same nucleotide sequence as the target gene (Integrated DNA technologies, USA) ) or the target gene, methylated DNA oligomer of SEQ ID NO: 6 (Integrated DNA technologies, USA) 1 μM, TMAC (Trimethylammonuim chloride) (Sigma aldrich, USA) 1 M and PCR amplification solution (Sisun Biomaterials, Korea) was added and mixed Then, a melting curve analysis was performed using a real-time gene amplifier (Real Time PCR machine, CFX96 ^TM Real Time PCR System, Bayrorad, USA). After reaction at 95 °C for 5 minutes, the temperature was lowered to 30 °C, and the temperature was increased by 1 °C from 30 °C to 90 °C, and dissolution curve analysis was performed while measuring fluorescence. The sequences used in the experiment are shown in Table 1 below.

The melting curve analysis result according to Example 1 is shown in FIG. 2A.

[Comparative Example 1]. Analysis of the melting curve of genes using Abasic PNA probes

A melting curve analysis was performed in the same manner as in Example 1, except that TMAC was not used. The melting curve analysis result according to Comparative Example 1 is shown in FIG. 2A.

[Comparative Example 2-1]. Analysis of melting curves of genes using DNA probes

A melting curve analysis was performed in the same manner as in Comparative Example 1, except that the DNA probe of SEQ ID NO: 1 was used. The melting curve analysis result according to Comparative Example 2-1 is shown in FIG. 2B.

[Comparative Example 2-2]. Analysis of melting curves of genes using DNA probes

A melting curve analysis was performed in the same manner as in Example 1, except that the DNA probe of SEQ ID NO: 1 was used. The melting curve analysis result according to Comparative Example 2-2 is shown in FIG. 2B.

[Comparative Example 3-1]. Analysis of the melting curve of genes using PNA probes

A melting curve analysis was performed in the same manner as in Comparative Example 1, except that the PNA probe of SEQ ID NO: 2 was used. The melting curve analysis result according to Comparative Example 3-1 is shown in FIG. 2C.

[Comparative Example 3-2]. Analysis of the melting curve of genes using PNA probes

A melting curve analysis was performed in the same manner as in Example 1, except that the PNA probe of SEQ ID NO: 2 was used. The melting curve analysis result according to Comparative Example 3-2 is shown in FIG. 2C.

[Comparative Example 4-1]. Analysis of the melting curve of genes using mismatched PNA probes

A melting curve analysis was performed in the same manner as in Comparative Example 1, except that a mismatch PNA probe of SEQ ID NO: 3 was used. The melting curve analysis result according to Comparative Example 4-1 is shown in FIG. 2D.

[Comparative Example 4-2]. Analysis of the melting curve of genes using PNA probes

A melting curve analysis was performed in the same manner as in Example 1, except that the mismatched PNA probe of SEQ ID NO: 3 was used. The melting curve analysis result according to Comparative Example 4-2 is shown in FIG. 2D.

[Example 2]. Methylation Detection Using Abasic PNA Probe and Methyl-affinity Molecules Following Restriction Enzyme Addition

As a non-methylation control DNA sample, the DNA sample and methylated DNA sample extracted from the Hela cell line (NEB, USA), which is known to not methylate the nucleotide sequence portion of the CpG island of the MGMT gene, is a representative glioblastoma cell line in which the gene is methylated. Experiments were conducted using DNA extracted from U-87 (ATCC, USA). Methylation analysis PCR using 10ng of gDNA isolated from the cell line, respectively, amplification solution with or without MSRE (methylation specific restriction enzyme) HhaI (Sissun Biomaterials, Korea) and PNA probe SEQ ID NO: 2 or 4 (CFX-96 Real-time) was performed.

PCR amplification conditions are as follows. After 15 minutes at 37°C and 5 minutes at 95°C, a total of 45 times were performed: 30 seconds at 95°C, 30 seconds at 56°C, and 30 seconds at 58°C. ) was used. The conditions for asymmetric PCR are as follows. 10 μl PCR amplification solution, 1 uM forward primer (SEQ ID NO: 7), 0.1 uM reverse primer (SEQ ID NO: 8), 0.1 uM unmethylated specific abasic PNA probe (SEQ ID NO: 4), 10 ng gDNA, 1 M TMAC, restriction enzyme HhaI 2.5 Unit (NEB, USA) was added or not, distilled water was added so that the total volume was 20 μl, and real-time PCR was performed. The results of PCR are shown in Fig. 3a.

In the case of methylated DNA extracted from the U-87 cell line (upper graph), no difference in amplification efficiency was confirmed depending on the addition of restriction enzymes, but in the case of DNA extracted from unmethylated cell lines, the difference in ΔC _t values was observed according to the addition of restriction enzymes. 9, which was 1.8 times higher than that of 5 when the general PNA probe according to Comparative Example 5 was used, confirming that the difference in amplification efficiency was significantly increased.

[Comparative Example 5]. Methylation detection using general PNA probes and methyl-affinity molecules following addition of restriction enzymes

PCR was performed in the same manner as in Example 2, except that 0.1 uM PNA probe (SEQ ID NO: 2) was used instead of 0.1 uM unmethylated specific abasic PNA probe (SEQ ID NO: 4). The result is shown in Fig. 3b.

In the case of methylated DNA extracted from the U-87 cell line (upper graph), no difference in amplification efficiency was confirmed depending on the addition of restriction enzymes, but in the case of DNA extracted from unmethylated cell lines, the difference in ΔC _t values was observed according to the addition of restriction enzymes. 5 is shown. This is a much lower value compared to Example 2 using a non-methylation-specific PNA probe.

[Experimental Example 1]. Sensitivity test of methylation detection method according to probe type

U-87 gDNA in which the cytosine of the target site of the PNA probe was determined to be 95% or more methylated was compared with Hela gDNA, a control sample in which the cytosine of the target site was determined to be 0% methylated, and 0.5%, 1%, 5%, 10 Methylation analysis PCR (CFX-96 Real-time) using DNA probes, general PNA probes, and non-methylation specific abasic PNA probes targeting U-87, Hela gDNA mixed or unmixed at %, 25%, 50% was carried out.

PCR amplification conditions are the same as in Example 2, and asymmetric PCR conditions are as follows. 10 μl PCR amplification solution, 1 uM forward primer (SEQ ID NO: 7), 0.1 uM reverse primer (SEQ ID NO: 8), 0.1 uM PNA probe (SEQ ID NO: 2) or 0.1 uM unmethylated specific PNA probe (SEQ ID NO: 4) or 0.1 After mixing uM DNA probe (SEQ ID NO: 3), 10ng gDNA, 1M TMAC, and 2.5 units of restriction enzyme HhaI, distilled water was added so that the total volume was 20 μl, and real-time PCR was performed.

The C _t values according to the PCR are shown in Table 2 below.

In the case of 1%, 0.5%, and 0% methylation sections of the general PNA and DNA probes indicated by the shades, since the Ct difference was not confirmed, it means that methylation detection is not possible in the target gene including 5% or less methylated genes. That is, it was confirmed that the detection limit was about 5% when using the general PNA and DNA probes.

On the other hand, it was confirmed that, when the detection method according to the present invention using a non-methylated specific PNA probe was used, it was possible to detect up to a sample containing 0.5% methylated DNA.

4 is a graph showing the experimental results, methylation detection performance and sensitivity through a ratio-specific combination of DNA isolated from standard cells (Hela) in which the MGMT gene is not methylated and U-87 cells in which MGMT is about 95% methylated. can be checked.

Although preferred embodiments of the present invention have been described above, the present invention is not limited thereto, and it is possible to carry out various modifications within the scope of the claims, the description of the invention and the accompanying drawings, and this also It is natural to fall within the scope.

Claims (12)

treating a target gene capable of methylation with a probe capable of specifically binding to a base of an unmethylated gene;
treating the target gene with a methyl-affinity molecule;
performing PCR (Polymerase Chain Reaction) on the target gene; and
Analyzing the melting curve by the PCR, calculating at least one of ΔT _m (melting temperature) and ΔC _t (cycle threshold); Containing, Method for detecting methylation of a gene using a non-methylated gene probe
(the ΔT _m = T _{m (target gene)} - T _{m (unmethylated gene consisting of the same nucleotide sequence as the target gene)} , and the ΔC _t = C _{t (target gene)} - C _{t (the same nucleotide sequence as the} target gene) _{is an unmethylated gene consisting} of). The method of claim 1,
Prior to treating the target gene with the probe, treating the target gene with a restriction enzyme to selectively cut the unmethylated site; further comprising, a method for detecting gene methylation. 3. The method of claim 2,
The restriction enzymes are HgaI, HhaI, Hinp1I, HpaI, SacII, AccII, Ava, BssHII, BstUI, HpaII, AatII, AciI, AcII, AfeI, AscI, Agel, BceAIBmgBI, BsaAI, BsaHI, BsiEI, BspDI, BstBI, ClaI, BstBI, HaeII, KasI, NaeI, NarI, NgoMIV, NotI, NruI, Nt.BsmAI, PaeR7I, PluTI, PmlI, PvuI, RsrII, SacII, SalI, SfoI, SgrAI, SmaI, SnaBI, SrfI, TspMI, ZraI, or a combination thereof , a method for detecting gene methylation. The method of claim 1,
The methyl-affinity molecule is tetramethyl ammonium (TMA), a method for detecting methylation of a gene. The method of claim 1,
The probe is any one or two or more selected from the group consisting of oligonucleotides, Locked Nucleic Acid (LNA), Peptide Nucleic Acid (PNA), and mixtures thereof, gene methylation detection method. The method of claim 1,
The method for detecting methylation of a gene, wherein the probe is a modified PNA probe that does not include a base at a position complementary to the methylation base of the target gene. The method of claim 1,
The methylation of the gene is a methylation detection method of a gene, including a methylated nucleotide sequence, a hydroxymethylated nucleotide sequence, a formyl methylated nucleotide sequence, a carboxyl methylated nucleotide sequence, or a combination thereof. The method of claim 1,
The detection method includes standard PCR, real time PCR, digital PCR, isothermal PCR, methylation specific PCR, real time methylation specific PCR, mass PCR , DNA chip, DNA FISH (Fluorescence in situ hybridization) any one or more selected from the, gene methylation detection method. The method of claim 1,
A method for detecting methylation of a gene, wherein the methylation detection limit of the target gene is less than 1%. The method of claim 1,
Wherein the probe is labeled with a reporter, a quencher, an intercalating fluorescent substance, or a combination thereof. The method of claim 1,
After performing the PCR, sequencing analysis step of the methylated target gene; further comprising, a gene methylation detection method. A kit for use in the method for detecting methylation of a gene according to any one of claims 1 to 10, comprising a probe capable of specifically binding to an unmethylated base of a target gene capable of methylation.

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