KR101553870B1 - Method of PCR-based DNA methylation analysis using DNA demethylase - Google Patents

Abstract

The present invention relates to a method for analyzing the degree of methylation of a target DNA, comprising the step of treating a target DNA extracted from a biological sample with DNA demethylase and then performing a DNA amplification reaction to detect an amplification product And (a) separating a specific fragment of target DNA extracted from the biological sample; (b) treating the DNA fragment of step (a) with DNA demethylase and performing a DNA amplification reaction to detect an amplification product; (c) detecting the amplification product by performing a DNA amplification reaction on the fragment of step (a) without treating the DNA demethylase; And (d) comparing the amplification products of step (b) and step (c).

Description

[0001] The present invention relates to a method for DNA methylation analysis based on PCR using DNA demethylase,

The present invention relates to a PCR-based DNA methylation analysis method using DNA demethylase, and more particularly, to a DNA methylation analysis method using DNA demethylase (hereinafter, DNA methylase) And detecting an amplification product; and (a) separating a specific fragment of a target DNA extracted from the biological sample; (b) treating the DNA fragment of step (a) with DNA demethylase and performing a DNA amplification reaction to detect an amplification product; (c) detecting the amplification product by performing a DNA amplification reaction on the fragment of step (a) without treating the DNA demethylase; And (d) comparing the amplification products of step (b) and step (c).

Although mutations that change the base in general genetic point of view are important phenomenon, in posterior sex, the expression pattern of gene changes by methylation which methyl group attaches to C5 position of cytidine (C) pyrimidine ring. This reaction occurs only when a guanine (G) comes after cytosine (C) (CpG), mediated only by the DNA methyltransferase enzyme, and S-adenosyl-methionine acts as a major methyl group. In this arrangement, cytosines tend to be methylated, and in the human genome, 3-5% of all cytosines are methylated.

These CpGs have been gradually reduced in evolution, and the degree and pattern of methylation of CpG present in the genome varies from species to species and varies very differently depending on the tissue. In the mammalian sequence, there is a region called 'CpG island' in which CpG is dense, and this region is closely related to the genomic gene of about 0.5 to 4 kb. This CpG island is located near the promoter that regulates transcription of the gene, but most CpG islands are not methylated. Therefore, if the CpG island of the promoter region that initiates the expression of a specific gene is methylated, the expression of the specific gene can be inhibited, which is important.

DEMETER (DME, DNA demethylase) is a representative DNA demethyl gene for plants that remove DNA methylation, and plays an important role in seed development and gene imprinting. In addition, the DME protein has a DNA glycosylase activity and a single-strand break (DNA strand break) to specifically recognize and remove 5-methylcytosine present in DNA regardless of the base sequence. SSB), and the DME enzyme has been developed to maximize the stability and efficiency of protein through engineering improvement for in vitro reaction optimization.

Currently, a variety of methods for analyzing DNA methylation, one of the major epigenetic mechanisms controlling eukaryotic chromatin structure and gene expression, have been developed and used, but there is no method with both accuracy and ease of analysis . Although there is a method of using methylation-sensitive restriction enzyme (MSRE) that is specific for DNA methylation as a method for rapid and simple analysis of DNA methylation, MSRE contains only the DNA methylation of a specific nucleotide sequence There was a problem.

In the present invention, DNA methylase (demethylase), which has an activity to induce single strand break (SSB) by specifically acting on methylated DNA regardless of the base sequence, is more easily and accurately DNA methylated And to provide a PCR-based technology capable of analyzing the PCR.

Korean Patent No. 0735045 discloses a method for measuring DNA methylation using a mouse B1 sequence and a method for assaying the anticancer activity of an anticancer drug using the method. Korean Patent No. 0801453 discloses a method for detecting various types of cancer DNA using the promoter methylation However, PCR-based DNA methylation analysis methods using DNA demethylase as in the present invention have not been disclosed.

SUMMARY OF THE INVENTION The present invention has been made in view of the above-described needs, and the present inventors have conducted intensive studies on the FWA gene, which is a flowering inhibitory gene of Arabidopsis thaliana, by combining DNA methylation level with DNA demethylase which is non- It was fast, accurate, and easy to measure. Through the above method, a sample treated with a DNA demethylase (DNA demethylase) and a sample not treated with the target DNA fragment extracted from an animal or a plant are amplified for a predetermined time, and then the amount of DNA amplification product is compared, The present inventors have completed the present invention by confirming that the DNA methylation level is high when the difference is large, and thus it is possible to select the epigenetic molecular marker existing in an animal or a plant.

In order to solve the above problems, the present invention provides a method for detecting a target DNA (target DNA) comprising the step of treating DNA target DNA extracted from a biological sample with DNA demethylase The degree of methylation.

(A) separating a specific fragment of a target DNA extracted from a biological sample; (b) treating the DNA fragment of step (a) with DNA demethylase and performing a DNA amplification reaction to detect an amplification product; (c) detecting the amplification product by performing a DNA amplification reaction on the fragment of step (a) without treating the DNA demethylase; And (d) comparing the amplification products of step (b) and step (c).

The present invention can provide a PCR-based technology capable of analyzing DNA methylation more easily and accurately by utilizing DNA demethylase that specifically functions on methylated DNA regardless of the base sequence, And can be used for the screening of epigenetic molecular markers. Especially, by diagnosing whether or not methylation of a specific promoter part is diagnosed, it can be useful for diagnosing or predicting cancer occurrence or cancer occurrence in a specific tissue.

Figure 1 shows the effect of DNA demethylating enzyme on the proportion of methylated DNA fragment. No M.SssI: M.SssI represents M.SssI DNA methylase-untreated sample: M.SssI DNA methylase-treated sample.
Figure 2 shows the effect of DNA demethylase on the proportion of methylated DNA fragments by the enzyme. Higher methylated DNA ratios require longer cycles for PCR amplification.
Figure 3 shows the degree of treatment of DNA demethylating enzyme (600ng) over time. The upper part is electrophoresis image, and each of them is quantified as FWA wild type (A), FWA wild type (B), and fwa posterior variant (C). The vertical axes of (A), (B) and (C) indicate the degree of methylation.
FIG. 4 shows the results of analysis of the DNA methylation level of the FWA gene, a flowering-inhibitory gene of Arabidopsis thaliana, in combination with DNA demethylase and real-time PCR. The flowering time differs depending on the degree of methylation of the FWA gene promoter region. When allele with low methylation is homozygote or heterozygote, delay of flowering time is observed. E of flowering means early flowering, and L means late flowering. In the genotype, + indicates the FWA wild type (Col-0), and - indicates the result of analysis of the fwa-bearing variant (epimutant, derived from Ler) by the CAPS marker. Ler (+ / +) has an early flowering phenotype like Col-0 wild type, but it has other genotype, namely SNP. In methylation, Y and N indicate the presence or absence of methylation of the FWA promoter region (bottom graph).

In order to accomplish the object of the present invention, the present invention provides a method for detecting an amplification product, comprising the step of treating a target DNA extracted from a biological sample with a DNA demethylase and performing a DNA amplification reaction to detect an amplification product A method for analyzing the degree of methylation of a target DNA is provided.

In the method of analyzing the degree of methylation of the target DNA of the present invention, the target DNA (target DNA) extracted from the biological sample is first treated with DNA demethylase, Although it depends on the amount of enzyme, preferably 500 ng of target DNA can be treated with 600 ng of DNA demethylase for 2 hours, but it is not limited thereto. Then, the target DNA subjected to DNA demethylase treatment is subjected to DNA amplification reaction and the amplification product is detected. When a relatively large amount of the amplification product is detected, it means that the methylation of the target DNA is decreased, If it is detected less, it means that the target DNA is methylated much. Therefore, the degree of methylation of the target DNA can be analyzed according to the relative amount of the amplification product.

The term " methylation "of the present invention means that a methyl group is added to the 5th carbon of the cytosine of DNA.

In the method for analyzing the degree of methylation of a target DNA according to an embodiment of the present invention, the biological sample may be animal or plant-derived, and preferably plant-derived, but is not limited thereto.

In the method for analyzing the degree of methylation of a target DNA according to an embodiment of the present invention, the target DNA may be a region including a "CpG island ", preferably a promoter region, but is not limited thereto.

The term "CpG island" in the present invention refers to a plurality of CG dinucleotides which are distinguished from CG pairs found in the genome. Here, C (Cytosine) is generally methylated, and since methyl-C is highly likely to be converted to T, CpG in the genome should be observed more often than C and G independent. However, many CpGs can be found because the methylation process must be suppressed in the vicinity of the promoter and in the start region of many genes. These regions are called CpG islands and typically range from a few hundred to several thousand base lengths.

In the present invention, the term "promoter " refers to a site where a RNA polymerase and various transcription-related regulatory elements bind to each other for quantitative regulation of gene expression for gene transcription and expression.

In the method for analyzing the degree of methylation of a target DNA according to an embodiment of the present invention, the DNA amplification reaction may be amplified by polymerase chain reaction (PCR) or any other suitable method for amplifying a nucleic acid molecule known in the art . Among them, PCR is a method of amplifying a target nucleic acid from a pair of primers that specifically bind to a target nucleic acid using a polymerase. Such a PCR method is well known in the art, and a commercially available kit may be used, and most preferably, it may be a real-time PCR, but is not limited thereto.

In the method for analyzing the degree of methylation of target DNA according to an embodiment of the present invention, detection of the amplification product can be performed by capillary electrophoresis, DNA chip, gel electrophoresis, radioactive measurement, fluorescence measurement or phosphorescence measurement , But is not limited thereto. As a method of detecting the amplification product, gel electrophoresis can be performed, and gel electrophoresis can be performed using acrylamide gel electrophoresis or agarose gel electrophoresis according to the size of the amplification product. In addition, capillary electrophoresis can be performed. Capillary electrophoresis, for example, can use the ABi Sequencer. Also, in the fluorescence measurement method, Cy-5 or Cy-3 is labeled at the 5'-end of the primer. When PCR is carried out, the target is labeled with a fluorescent label capable of detecting the target sequence. The labeled fluorescence is measured using a fluorescence meter can do. In addition, in the case of performing the PCR, the radioactive isotope such as ³² P or ³⁵ S is added to the PCR reaction solution to mark the amplification product, and then a radioactive measurement device such as a Geiger counter or liquid scintillation counter The radioactivity can be measured using a liquid scintillation counter.

In addition,

(a) separating a specific fragment of target DNA extracted from a biological sample;

(b) treating the DNA fragment of step (a) with DNA demethylase and performing a DNA amplification reaction to detect an amplification product;

(c) detecting the amplification product by performing a DNA amplification reaction on the fragment of step (a) without treating the DNA demethylase; And

(d) comparing the amplification products of step (b) and step (c).

In step (d), amplification products of steps (b) and (c) are compared, and if there is a difference in the amount of amplification product, Specific fragments can be predicted with a progeny genetic molecular marker, and without differences, a particular fragment can be predicted to be not a progeny genetic molecular marker.

In other words, since the epigenetic molecular marker is usually a site where methylation occurs frequently, the DNA amplification reaction is performed without treating the DNA demethylase because the cleavage is caused by the DNA demethylase and the amount of the amplification product is small, And the amount of amplified product is different. However, methylation does not occur at sites other than the epigenetic molecular marker, so DNA amplification reactions may be performed with or without the DNA demethylase enzyme. Based on this principle, it is possible to select epigenetic molecular markers.

In the method for screening the epigenetic molecular marker according to an embodiment of the present invention, the DNA amplification reaction and the detection of the amplification product are as described above.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Experimental Method

Artificially methylated DNA To DNA Demethylase  After processing PCR  Compare the degree of amplification

The FWA promoter genomic DNA of Arabidopsis thaliana was amplified by PCR amplification, and the prepared DNA was divided into two tubes. One of them was treated with M.SssI DNA methyltransferase to methylate CpG to cytosine (Sample A) One did not process the enzyme (Sample B). Differential DNA methylation samples were prepared by mixing samples A and B at a ratio of 100%, 75%, 50%, 25%, 0%. The treatment conditions of DME DNA demethylase for each DNA sample are as follows. DNA demethylation reaction was performed using 1X Tris buffer (10 mM Tris-HCl, pH 7.4, 50 mM NaCl, 0.5 mM DTT) and the DNA demethylase enzyme was MBP-DMEΔN677ΔIDR1 :: lnk protein (Mok et al., 2010, PNAS 107: 200 ng of DNA was treated with 300 ng of DME enzyme in 20 μl of the reaction solution at 37 ° C for 1 to 2 hours and enzyme inactivation was performed at 65 ° C. Was diluted 1/1000, and real-time PCR was performed. PCR was performed using a pair of primers capable of amplifying a portion having no CpG as a control and a portion capable of amplifying a portion having a difference in the degree of DNA methyl, and the amplification value of the control group was set to 1 to amplify a portion having CpG Value.

Actual differential DNA  Possible epigenetic applications for samples showing methylation Molecular marker  Development

In this experiment, FWA, a flowering inhibitor of Arabidopsis thaliana, was used as a target to identify actual differential DNA methylation. The wild-type Arabidopsis FWA gene is suppressed by promoter methylation, but fwa mutants are epigenetic mutants whose expression is induced by the diminution of DNA methylation and ultimately delayed flowering time. DNA of Arabidopsis wild type ( Ler , Col - 0) and fwa - 1 ( Ler ) mutants were extracted using CTAB method. 500ng of gDNA was treated with 600ng of DME for 2 hours, and the FWA gene promoter region was amplified by real-time PCR using DME-treated DNA as a template. The relative amplification of the FWA promoter region was determined for each sample by setting the amplification level of control ASA1 (a gene having no methylation at all) to 1. We analyzed the correlation between phenotype, genotype and DNA methylation using wild type (FWA / FWA), fwa mutant (fwa / fwa), F1 hybrid (FWA / fwa) and F2 group isolated here. The flowering time of the wild type ( Col- 0 , Ler ) and mutant (fwa-1) and the first and second generations of Arabidopsis thaliana was measured.

To confirm the hybridization between Col- 0 and fwa- 1 ( Ler ), CAPS markers (Cleaved Amplified Polymorphic Sequences) were constructed using SNPs present in the FWA promoter region, and wild-type, mutant, Genotypes of the second generation were identified.

Example  1. Differential DNA  To the methylated sample DNA Demethylation  After enzyme treatment PCR  Comparison of amplification degree

As the ratio of methylated cytosine-containing DNA fragments was increased by M.SssI, the DNA template cleavage by DNA demethylase caused a lot of PCR templates. Therefore, it was observed that the amplification of PCR was slowed due to the effect of DNA demethylase as the proportion of DNA fragment containing 5-methylcytosine was larger (FIG. 1). In addition, the DNA methylation level could be determined by comparing the amplification curve by DNA demethylase treatment (Fig. 2).

Example  2. DNA methyl  Optimal for level analysis DNA Demethylase  Understand reaction conditions

The minimal processing time and the minimum amount of DNA demethylating enzyme were determined using the differences in DNA methylation. 500 ng DNA-free enzyme-free, 1 hour, 2 hour, and 3 hour treatment groups. The FWA promoter region showing the difference in DNA methylation was used as a template for each of the PCR products. There was no significant difference between the groups treated with DNA demethylase for 2 hours and 3 hours. However, when treated with DNA methylase for 2 hours and 3 hours, there was a significant difference. In this way, it was found that treating 500ng of DNA of Arabidopsis with 600ng of DNA demethylating enzyme for 2 hours is the most suitable state for obtaining the desired result (FIG. 3).

Example  3. Differential DNA  That can distinguish methylation Epigenetic Molecular marker  Development

Using the second generation of wild type and mutant and hybridized offspring of different Arabidopsis species, we correlated phenotype, genotype and progeny genotype. It was confirmed that the second generation of the offspring showing early flowering as in wild type coincided with the genotype wild type ( Col- 0 ), and the PCR result after DNA demethylating enzyme treatment was also consistent (Fig. 4). On the other hand, it was confirmed that the second generation of offspring showing late flowering, such as mutation type and hybridization one, were genotype mutant or hybrid, and PCR results were also consistent. Therefore, PCR analysis using DNA demethylase was very effective in identifying genes with high DNA methylation, and it could be used as a molecular genetic marker.

Claims (9)

DNA methylase (DNA demethylase), which has activity to induce single-strand breakage by acting specifically on methylated DNA regardless of the base sequence, is applied to target DNA extracted from biological sample The DNA methylation of the target DNA was judged to be decreased when the amount of the amplified product was detected at a similar level to the sample not treated with the DNA demethylase enzyme and the DNA methylation enzyme was amplified And determining that methylation of the target DNA is increased if the amount of the product is relatively small. The method according to claim 1, wherein the biological sample is animal or plant-derived. 2. The method according to claim 1, wherein the target DNA is a promoter region. The method according to claim 1, wherein the DNA amplification reaction is a PCR (polymerase chain reaction). 5. The method according to claim 4, wherein the PCR is a real-time PCR. The method according to claim 1, wherein the detection of the amplification product is performed by gel electrophoresis, capillary electrophoresis, DNA chip, radioactivity measurement, fluorescence measurement or phosphorescence measurement. (a) separating a specific fragment of target DNA extracted from a biological sample;
(b) treating the DNA fragment of step (a) with a DNA demethylase that specifically acts on the methylated DNA to induce truncation of the strand, regardless of the nucleotide sequence, Performing a reaction to detect an amplification product;
(c) a step of amplifying a DNA fragment of the step (a) without treating the DNA demethylase, which has an activity of specifically inducing single-strand breakage by acting on the methylated DNA regardless of the nucleotide sequence, Performing a reaction to detect an amplification product; And
(d) comparing the amplification products of step (b) and step (c). [8] The method according to claim 7, wherein step (d) comprises comparing amplification products of steps (b) and (c) Wherein the specific fragment is predicted to be a non-epigenetic molecular marker if it is not present. 8. The method of claim 7, wherein the DNA amplification reaction is a real-time PCR (polymerase chain reaction).

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