KR101561034B1 - Method for Methylation Detection of Converted DNA by Bisulfite-Treatment Using Inosine-containing Modified Primers - Google Patents

Abstract

The present invention relates to a method for detecting methylation using an inosine-containing primer. When methylation-specific primers containing the inosine residue according to the present invention are used, the specificity to the target sequence is higher than that of the conventional methylation-specific primers, and there is no loss in sensitivity, so that even minute amounts of methylated DNA can be efficiently detected have.

Description

METHOD FOR Methylation Detection of Converted DNA by Bisulfite-Treatment Using Inosine-Containing Modified Primers [

The present invention relates to a method for detecting methylation of transformed DNA by bisulfite treatment using inosine-containing modified primers and a kit for detecting methylation containing the primers.

Cytosine methylation is a chemical change that occurs on DNA without changes in the sequence due to epigenetic changes that affect normal gene expression. There is a cluster in which a CG dinucleotide called CpG island is concentrated at a high density in a transcriptional regulatory region including a promoter of a specific gene or a 5 'untranslated region, CpG island cytosine (C) is converted to 5-methylcytosine by methylation at the 5th carbon position by DNA methyltransferase after DNA synthesis. This methylation phenomenon is known to be a cause of various diseases including cancer, and it is known that methylation of CpG island inhibits the expression of the corresponding gene. In particular, it is known that CpG islands of the tumor suppressor gene are methylated and their expression is blocked, resulting in cancer. DNA methylation has been recognized as an optimal tool for the early detection of cancer since it is a typical phenomenon that occurs at the earliest stages of cancer development. Recently, the development of a diagnostic technique using a methylation phenomenon of a specific gene as a biomarker for early diagnosis and screening of cancer has been actively studied. Methods such as methylation-specific PCR (hereinafter referred to as MSP) or automatic DNA sequencing have been actively attempted to detect CpG island methylation associated with such biomarker genes.

In order to maximize the accuracy of cancer diagnosis using the methylation of a specific gene, to analyze the development of cancer at each step, and to distinguish between cancer and aging changes, methylation of all cytosine bases in CpG islands There is a need for a method to analyze accurately. The orthodox method is to amplify the gene region containing CpG island by methylation - specific PCR (MSP) and to use the sequencing method (bisulfite genomic sequencing method) together. However, there is no way to accurately and rapidly analyze the various changes in the promoter methylation of many genes, which can be used to assess the various stages of cancer in diagnosis, initial diagnosis, or clinical trials.

Polymerase chain reaction (PCR) is the most effective method for selectively amplifying a specific DNA fragment. There are many kinds of PCR, but the "methylation specific PCR (MSP)", which is the basis of the present invention, is the most widely used method for detecting hypermethylation of CpG island. After treatment of bisulfite with DNA And amplifying the methylated DNA by PCR (FIG. 1A).

The position of 5-methylcytosine present in the DNA can not be identified by sequencing if the target DNA is not treated with bisulfite before the PCR, since 5-methylcytosine has the same base pairing behavior as cytosine Because. In addition, PCR amplification has the problem that epigenetic information retained by 5-methylcytosine is completely lost. On the other hand, when bisulfite is treated with DNA, the methylated cytosine of CpG island remains as cytosine and the unmethylated cytosine is converted to uracil. Therefore, primer pairs required for PCR are designed to be complementarily bound to methylated cytosines, which are complementary to at least two CpG dinucleotides, so that a specific methylation pattern among many methylation patterns can be sensitively detected. Therefore, when such a primer pair is added, amplification occurs only when methylated cytosine is present in the template DNA, and amplification does not occur in the absence of methylated cytosine. In contrast, it is also possible to use primers that amplify only non-methylated DNA. The MSP method is also applied to real-time PCR analysis using fluorescent probes or fluorescent dyes to enable detection of methylation with high throughput while quantitative analysis is possible, and sequencing of the methylated DNA sequence It is also possible.

However, even if a primer used in the MSP recognizes a specific methylation pattern and amplifies the DNA, it is also common to anneal and amplify a similar base sequence at a position other than the target sequence. This is called nonspecific amplification. If nonspecific amplification occurs, the PCR product may be generated even in the case of non-methylated DNA, and the specificity of detection of methylation is lowered. As a method for reducing nonspecific amplification in conventional PCR, there have been known methods for controlling the number of PCR cycles, controlling the concentration of primers, using high annealing temperature primers, and replacing some of the primer sequence base with an inosine base (I) found in tRNA anticodon (Ben-Dov E. et al., Appl. Environ Microbiol, 2006). Inosine is capable of base pairing with any of the bases (A, G, C, T, and U) and its bonding force is weaker than when complementary nucleotides are actually bonded. , It has been known that the specificity of a primer to a target DNA can be enhanced when some of the bases of the primer are replaced with inosine. However, there is no case in which an inosine-containing modified primer is used for MSP for methylation detection of transformed DNA after bisulfite treatment.

The present inventors have made intensive efforts to solve the above problems, and as a result, they have found that inosine-containing modified primers in which nucleotides of some of the nucleotide sequences of methylation-specific primers are replaced with inosine (I) by applying the feature of inosine to PCR or sequencing (FIG. 1B), it was finally confirmed that the methylated DNA converted after the bisulfite treatment can be detected with high specificity without loss of sensitivity, thereby completing the present invention.

It is an object of the present invention to provide a method for detecting methylation of DNA with increased specificity without loss of sensitivity.

In order to accomplish the above object, the present invention provides a method for producing DNA comprising the steps of treating bisulfite with DNA; And amplifying the transformed DNA using a primer and a DNA polymerase capable of amplifying the DNA transformed by the bisulfite treatment, wherein the primer comprises the DNA Of methylated cytosine and complementary to a sequence region comprising methylated cytosine of 1 to 5 inosine.

The invention also bisulfite converted SDC2 gene or its include methylated cytosine present in a CpG island associated with substituted with sequence portions and complementary to, the one to five bases of inosine, which SDC2 gene after treatment, or the Provides a primer pair for methylation detection of the associated CpG islands.

The invention also, SDC2 Treating the gene or an associated CpG island with bisulfite; And a primer pair in which 1 to 5 bases are substituted with inosine and SEQ ID NOS: 2 and 3, respectively, capable of amplifying the SDC2 gene or its associated CpG island converted by the bisulfite treatment, and DNA polymerase The converted SDC2 A method for detecting methylation of an SDC2 gene or an associated CpG isoform comprising the step of amplifying a gene or an associated CpG island.

The present invention also provides a DNA methylation detection kit comprising a primer and a DNA polymerase capable of amplifying DNA converted by a bisulfite treatment, wherein the primer comprises a sequence comprising methylated cytosine of the transformed DNA And wherein the kit contains 1 to 5 inosine.

The invention also bisulfite converted SDC2 gene or its include methylated cytosine present in a CpG island associated with substituted with sequence portions and complementary to, the one to five bases of inosine, which SDC2 gene after treatment, or the A primer pair for detecting methylation of an associated CpG island, and a kit for detecting methylation of an SDC2 gene or an associated CpG island comprising a DNA polymerase.

The modified methylation-specific primer containing the inosine residue according to the present invention has a higher specificity to the target sequence than the conventional methylation-specific primer and has no sensitivity loss, so that methylated DNA can be effectively detected have.

1 compares the principle of methylation specific PCR (A) using a common primer and methylation specific PCR (B) using an inosine primer.
FIG. 2 shows the results of serial dilution of unmethylated DNA corresponding to the nucleotide sequence of + 261 to +610 bp corresponding to 5 'UTR (untranslated region) of syndecan2 ( SDC2 ) gene from 10 ⁹ copies to 1 copy SDC2 PCR amplification using methylation-specific primer (A) and inosine-containing primer (B) of the gene.
Figure 3 compares the detection sensitivity of methylation specific primers and inosine primers to methylated DNA by real time PCR.
Figure 4 compares the real time PCR results of the methylation DNA of the methylation specific primer and inosine primer with the average Ct (cycle threshold).

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art. The bisulfite of the present invention refers to a bisulfite, which means the same material as the disulfite or hydrogen sulfite.

In the present invention, the detection specificity test of inosine-containing modified methylation-specific primers and the detection sensitivity test of inosine-containing modified methylation-specific primers using real-time PCR were performed. As a result, it was confirmed that the inosine-containing modified methylation-specific primer had a higher specificity and no sensitivity loss in methylation detection than a methylation-specific primer containing no inosine.

Accordingly, in one aspect, the present invention relates to a method for treating DNA comprising: treating bisulfite with DNA; And amplifying the transformed DNA using a primer and a DNA polymerase capable of amplifying the DNA transformed by the bisulfite treatment, wherein the primer comprises the DNA Of methylated cytosine and complementary to a sequence region comprising methylated cytosine of 1 to 5 inosine.

In one embodiment of the present invention, the above methylation specific PCR (MSP) was carried out to confirm that the inosine-containing modified primer specifically detected methylated DNA only: (a) CpG island of specific DNA Treating bisulfite to unmethylated bisulfite; (b) cloning the unmethylated nucleotide sequence of step (a) by a gene synthesis method and cloning it into a plasmid vector; (c) designing and synthesizing a methylation-specific amplification primer capable of annealing with the specific DNA of step (a); (d) replacing one or more nucleotides in the sequence of the primer of step (c) with inosine; And (e) amplifying the methylated DNA using a PCR instrument and then performing electrophoresis to confirm the PCR product.

In the above example, only one nucleotide was substituted with inosine, but it is not limited thereto, and one or more nucleotides can be substituted by a known inosine substitution method. In the above examples, in order to compare whether the inosine-containing modified methylation specific primers have superior specificity compared to the existing methylation specific primers, the unmethylated SDC2 Gene sequences were compared. When bisulfite is treated with DNA, the methylated cytosine of the CpG island remains intact, and the unmethylated cytosine is converted to uracil. As a result of the cross-reactivity comparison, PCR products were generated by nonspecific amplification despite the absence of cytosine in the CpG isoform of the target DNA, whereas the inosine-containing modified primer had no specific amplification, It showed excellent.

In the present invention, the detection method may be selected from the group consisting of PCR, DNA sequencing, and primer extension. However, the present invention is not limited thereto, (real time PCR). In addition, the real-time PCR may be characterized by using a probe labeled with a fluorescent reporter or a non-specific fluorescent dye.

In the present invention, PCR includes general PCR, methylation specific PCR, quantitative PCR including real time PCR, and digital PCR. DNA sequencing includes primers such as bisulfite sequencing, pyrosequencing, Sanger sequencing, hybridization sequencing, etc. A sequencing method, and the like, but the present invention is not limited thereto, and the PCR or DNA sequencing can be used without limitation as long as it is used in this technical field.

In one embodiment of the present invention, the real time PCR was performed to confirm the detection sensitivity of the inosine-containing modified methylation specific primers, including the following steps: (a) specific to methylated specific DNA Synthesizing a probe capable of hybridizing with the probe; (b) designing and synthesizing an inosine-containing modified methylation-specific amplification primer capable of annealing with a specific DNA; (c) amplifying the methylated DNA by real-time PCR using the probe of (a) and the primer of (b); And (c) measuring the cycle threshold (Ct) value of the PCR product.

The above real-time PCR is also called a quantitative polymerase chain reaction (qPCR). It is possible to amplify a target gene and quantitatively analyze it. The principle is the same as general PCR, but DNA amplified in real time during the reaction can be detected.

There are two commonly used methods in real-time PCR. The first is to treat non-specific fluorescent dyes such as Sybergreen by infiltrating into double stranded DNA and the second is to treat an oligonucleotide DNA probe that can specifically hybridize to a specific sequence . The probe is an oligonucleotide (10-100 bp short single stranded DNA or RNA), and has a radioactive substance or fluorescent reporter acting as a marker at the 5 'end. In the DNA amplification step, an exonuclease such as Taq polymerase The fluorescence is emitted from the fluorescent material. It is detected by X-ray, ultraviolet light, c-focal microscope, or antibody, depending on the type of fluorescent material that is penetrated into the DNA or labeled on the probe. Both methods make it possible to measure the amount of amplified DNA in real time.

The nonspecific fluorescent dyes include Sybergreen, Evagreen, Cy3, Cy5, biotin binding materials, Alexa 647, Alexa 555, (5- (2-aminoethyl) amino-1-naphthalene sulfate) , Tetramethylrhodamine (TMR), tetramethylrhodamine isocyanate (TMRITC), x-rhodamine, and Texas red. However, the present invention is not limited thereto. The fluorescent dyes can be used without limitation as long as they are used in this technical field.

In an embodiment of the present invention, a real-time PCR was performed using a TaqMan probe with a fluorescent reporter (fluorescent substance) of 6-carboxyfluorescein ( FAM ). When compared with the negative control using primers containing no inosine, Lt; RTI ID = 0.0 > methylation-specific primers. ≪ / RTI > Fluorophores that may be labeled may be selected from the group consisting of fluorescein, tetramethylrhodamine (TMR), Cy3, Cy5, and Texas red, but are not limited thereto. The fluorescent reporter can be used without limitation as long as it is used in this technical field.

In another aspect, the present invention provides a method for producing an SDC2 gene, which is complementary to a sequence region comprising a methylated cytosine present in a transformed SDC2 gene or an associated CpG island after bisulfite treatment, wherein 1 to 5 bases are substituted with inosine Or a pair of primers for detecting the methylation of the CpG islands associated therewith.

In the present invention, it may be characterized in that the primer pairs for detection of methylation of the SDC2 gene or CpG islands associated with 1 to 5 bases in SEQ ID NO: 2 and SEQ ID NO: 3 are substituted with inosine. Also, the primer pair may be a pair of primers for detecting the methylation of the SDC2 gene or the CpG islands associated therewith, as shown in SEQ ID NO: 4 and SEQ ID NO: 5.

In the present invention, "CpG islands associated therewith" means CpG islands located at sites including a promoter and 5'-UTR (untranslated region) necessary for SDC2 gene expression.

SDC2 The primers amplifying the gene or the associated CpG islands include all sequences in which 1 to 5 substitutions are made with the base inosine residue at any position in SEQ ID NO: 2 and SEQ ID NO: 3. The primers having the sequences of SEQ ID NOS: 4 and 5 are those in which the fourth base from the 3 'end of the primer having the sequence of SEQ ID NO: 2 and SEQ ID NO: 3 is substituted with inosine (I).

The invention in another aspect, SDC2 Treating the gene or an associated CpG island with bisulfite; And SEQ ID NO: 2 capable of amplifying the SDC2 gene or its associated CpG isoform transformed by the bisulfite treatment and 1 to 5 bases in SEQ ID NO: 3 being substituted with inosine, or represented by SEQ ID NO: 4 and SEQ ID NO: 5 Lt; / RTI > DNA polymerase and the transformed SDC2 < RTI ID = 0.0 > The present invention relates to a method for detecting methylation of an SDC2 gene or an associated CpG isoform comprising the step of amplifying a gene or an associated CpG island.

The transformed DNA to be amplified is human SDC2 Gene, and has a nucleotide sequence as shown in SEQ ID NO: 1 after the bisulfite treatment. SDC2 The primers amplifying the gene or its associated CpG islands include all sequences in which 1 to 5 substitutions are made to the inosine residues of the base at any position in SEQ ID NO: 2 and SEQ ID NO: 3. The primers having the sequences of SEQ ID NOS: 4 and 5 are those in which the fourth base from the 3 'end of the primer having the sequence of SEQ ID NO: 2 and SEQ ID NO: 3 is substituted with inosine (I).

In the present invention, the detection method may be selected from the group consisting of PCR, DNA sequencing, and primer extension. The PCR may be real-time PCR. In addition, the real-time PCR may be characterized in that a probe of SEQ ID NO: 6 or a non-specific fluorescent dye labeled with a fluorescent reporter is used. The probe of SEQ ID NO: 6 is a probe having a sequence capable of specifically hybridizing to the SDC2 gene.

In another aspect, the present invention relates to a primer capable of amplifying a DNA converted by a bisulfite treatment and a kit for detecting methylation of a DNA comprising a DNA polymerase, wherein the primer comprises a methylated It is complementary to a sequence region including cytosine, and can be characterized by containing 1 to 5 inosine.

In another aspect, the present invention relates to a method for producing an SDC2 gene or an SDC2 gene, which is complementary to a sequence region comprising a methylated cytosine of a transformed SDC2 gene or an associated CpG island after bisulfite treatment and wherein 1 to 5 bases are substituted with inosine A primer pair for detecting the methylation of CpG islands associated therewith, and a kit for detecting methylation of an SDC2 gene or an associated CpG isoform comprising DNA polymerase. Preferably 1 to 5 bases in SEQ ID NO: 2 and SEQ ID NO: 3 are substituted with inosine, or more preferably, primer pairs represented by SEQ ID NO: 4 and SEQ ID NO: 5 and SDC2 containing DNA polymerase Or a kit for detecting methylation of CpG islands associated therewith. It will be apparent to those skilled in the art that a pair of inosine-containing modified primers capable of amplifying DNA comprising a methylated CpG island may be provided for the kit for detecting methylation of the present invention, but not limited thereto .

The kit of the present invention comprises a partitioned carrier means for containing a sample, a first container capable of bisulfite treatment of the template DNA, a primer pair complementary to the 5'-CpG-3 'base sequence region for amplifying the sample , And a third container for detecting the amplified product.

The carrier means is suitable for containing one or more containers such as bottles, tubes, and each container contains the independent components used in the method of the present invention. In the context of the present invention, those skilled in the art will readily be able to dispense the necessary formulation in a container.

Methylation detection method

Methylation specificity PCR  ( 메틸레이션 specific PCR )

When biosulfite is treated with genomic DNA, the cytosine in the 5'-CpG'-3 region is left as it is when it is methylated, and when it is unmethylated, it changes into uracil. Therefore, a PCR primer corresponding to the 5'-CpG-3 'nucleotide sequence was prepared from the base sequence after bisulfite treatment. At this time, PCR primers corresponding to methylation and two types of primers corresponding to unmethylated primers were prepared. When genomic DNA is converted into bisulfite and then PCR is carried out using the above two types of primers, PCR products are produced by using primers corresponding to the methylated nucleotide sequence when methylated, and in the case of unmethylated PCR products are produced using primers corresponding to unmethylated sequences. The methylation can be qualitatively confirmed by agarose gel electrophoresis.

Real-time methylation specificity PCR  ( real time 메틸레이션 specific PCR )

Real-time methylation specific PCR is the conversion of methylation-specific PCR method to real-time measurement method. The PCR primer corresponding to the methylation of biosulfite treated with genomic DNA was designed and real-time PCR was performed using these primers . At this time, there are two methods of detecting using a TaqMan probe complementary to the amplified nucleotide sequence and using Sybergreen. Therefore, real-time methylation-specific PCR can quantitatively analyze only methylated DNA. At this time, in A standard curve was prepared using an in vitro methylated DNA sample. For normalization, genes without 5'-CpG-3 'sequences were amplified in the nucleotide sequence together with a negative control, and the degree of methylation was quantitatively analyzed.

Kit ( Kit )

According to the present invention, there is provided a kit useful for detecting methylation of DNA. The kit of the present invention comprises a partitioned carrier means for containing a sample, a first container capable of bisulfite treatment of the template DNA, a primer pair complementary to the 5'-CpG-3 'base sequence region for amplifying the sample , And a third container for detecting the amplified product.

The carrier means is suitable for containing one or more containers such as bottles, tubes, and each container contains the independent components used in the method of the present invention. In the context of the present invention, those skilled in the art will readily be able to dispense the necessary formulation in a container.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these embodiments. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Example  One: Inosine  Containing variant methylation-specific Primer  Detection specificity test

In order to confirm the detection specificity of the inosine-containing modified methylation-specific primer, bisulfite treatment was performed on the 5'UTR (corresponding to +261 to +610 bp from the transcription start point (+1)) of the human gene SDC2 The nucleotide sequence corresponding to the unmethylated state was prepared by a gene synthesis method and cloned into a TOPO pCR2.1 plasmid vector (Life Technologies) (Bioneer, Inc.).

Sequence 1: +261 to +610 bp

5'-aaattagaaa ttgaattttg gtatgggaaa ggagtttgtg gaggagtaaa attatagtag agtaagaaga gttttagaga gtagtttttt tggagtatta attttgtgtt gggagtgtag aaattaataa gtgagagggt gttgtgtttt tggggtgtag ttgtgggtgg tgggagtagg tgtaggagga ggaagtgagt gtttttgagt tttgagtttg agtttttgag tttgagttgt aattgttgtg gtattttgtt ttggatttgt gtgtgtgggt tgtgttgagt gttgggtagg aggttttgtt ttgttttggt tgtaagtagt ggttgggagt agttggtttt-3 '(SEQ ID NO: 1)

The methylation-specific primer for SDC2 gene amplification was designed using the MethPrimer program (http://www.mspprimer.org/cgi-mspprimer/design.cgi) and synthesized at Bioneer Inc.

SDC2-F: 5'-GTAGAAATTAATAAGTGAGAGGGC-3 '(SEQ ID NO: 2)

SDC2-R: 5'-ACGACTCAAACTCGAAAACTCG-3 '(SEQ ID NO: 3)

The inosine-containing SDC2 gene methylation-specific primer was synthesized in biona by replacing one nucleotide in the above primer sequence with inosine (I).

SDC2-FIP: 5'-GTAGAAATTAATAAGTGAGA I GGC-3 '(SEQ ID NO: 4)

SDC2-RIP: 5'-ACGACTCAAACTCGAAAA I TCG-3 '(SEQ ID NO: 5)

In addition, TaqMan probe for the detection of amplified products in real time PCR was synthesized from bioneer.

SDC2-probe: 5'-FAM-TTCGGGGCGTAGTTGCGGGCGG-3 '(SEQ ID NO: 6)

In order to compare the detection specificity between inosine-containing modified methylation-specific primers and general methylation-specific primers, non-methylated SDC2 And the presence or absence of cross-reactivity with the gene sequence (Fig. 2). For this purpose, the unmethylated SDC2 plasmid DNA was diluted 10-fold in succession from 10 ⁹ copies to 1 copy. These diluted plasmids were used as templates and amplified using a Rotor-Gene Q PCR instrument (Qiagen) with an inosine-containing modified methylation-specific primer and a general methylation-specific primer. PCR was carried out at 95 ^° C for 5 minutes, and the PCR reaction solution (2 μl; 5 × AptaTaq DNA Master (Roche Diagnostics), 4 μl; PCR primer, 2 μl (2 pmole / μl) After the treatment, it was performed 40 times for 15 seconds at 95 ^° C and 1 minute at 61 ^° C. The PCR product was electrophoresed on 1% agarose gel to confirm the PCR product.

As a result, as shown in FIG. 2A, when a methylation-specific primer was amplified using inosine-containing modified methylation-specific primers, nonspecific amplification in which a PCR product was produced when 10 ⁵ copies or more of unmethylated DNA was present Nonspecific amplification was not observed even in the presence of 10 ⁹ copies of unmethylated DNA (FIG. 2B). From these results, it was confirmed that the specificity of inosine-containing modified methylation-specific primers was excellent in the detection of methylation.

Example  2: Real time PCR Using Inosine  Containing variant methylation-specific Primer  Detection sensitivity test

Real time PCR was performed to compare the detection sensitivity of inosine-containing modified methylation-specific primers with those of normal methylation-specific primers. For this, a probe (SEQ ID NO: 6) capable of specifically hybridizing to the methylated SDC2 gene synthesized in Example 1 was used.

20 ng to 50 pg of the genomic DNA of the colon cancer cell line HCT116 (Korea Cell Line Bank KCLB No. 10247) in which the SDC2 gene was completely methylated was added to 20 ng of the unmethylated human leukocyte cell genome DNA (BioChain, Cat. No. D1234148) Followed by continuous dilution and mixing. The genomic DNA was treated with bisulfite using EZ DNA methylation-Gold kit (Zymo Research, USA), and then eluted with 10 μl of sterilized distilled water and subjected to methylation-specific real time PCR (qMSP) Respectively. Real time PCR was carried out using the general methylation specific primers (SEQ ID NO: 2 and SEQ ID NO: 3) synthesized in Example 1 and inosine-containing modified methylation specific primers (SEQ ID NO: 4 and SEQ ID NO: 5) . Real-time PCR was performed using a Rotor-Gene Q PCR instrument (Qiagen). 2 μl (2 pmole / μl), 2 μl (2 pmole / μl) of DW primer, 2 μl (2 pmole / μl) of the PCR reaction solution (template DNA, PCR was performed at 95 ^° C for 5 minutes, followed by 40 cycles of 95 ^° C for 15 seconds and 61 ^° C for 1 minute. The amplification of the PCR product was confirmed by measuring the cycle threshold (Ct) value. Real time PCR per dilution concentration was repeated 9 to 24 times.

As a result, as shown in FIG. 3, methylation detection was 100% regardless of primers at a concentration of 200 pg or more when the continuously diluted HCT116 was detected. However, at 100 pg and 50 pg, the inosine- 24 in 24 rounds), but the overall methylation-specific primers showed a low detection rate of 95.8% (23 times in 24 rounds) and 79.2% (19 rounds in 24 rounds), respectively (Figure 3). The PCR product was electrophoresed on 1% agarose gel to confirm the PCR product. As a result, it was confirmed that the detection sensitivity of the inosine-containing modified methylation-specific primer was better.

Figure 4 shows the average of the Ct values measured in the experiment. Real-time PCR Ct values using inosine-containing modified primers at all concentrations tested were lower than in the case of using conventional primers, indicating that the amplification performance of the inosine-containing modified primers was better. This difference in Ct value was found to be greater as the concentration of methylated DNA was lower.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will readily appreciate that many modifications are possible in the exemplary embodiments without materially departing from the novel teachings and advantages of this invention. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Genomictree, Inc. <120> Method for Methylation Detection of Converted DNA          Bisulfite-Treatment Using Inosine-containing Modified Primers <130> P14-B091 <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 350 <212> DNA <213> Homo Sapiens <400> 1 aaattagaaa ttgaattttg gtatgggaaa ggagtttgtg gaggagtaaa attatagtag 60 agtaagaaga gttttagaga gtagtttttt tggagtatta attttgtgtt gggagtgtag 120 aaattaataa gtgagagggt gttgtgtttt tggggtgtag ttgtgggtgg tgggagtagg 180 tgtaggagga ggaagtgagt gtttttgagt tttgagtttg agtttttgag tttgagttgt 240 aattgttgtg gtattttgtt ttggatttgt gtgtgtgggt tgtgttgagt gttgggtagg 300 aggttttgtt ttgttttggt tgtaagtagt ggttgggagt agttggtttt 350 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> SDC2-F <400> 2 gtagaaatta ataagtgaga gggc 24 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> SDC2-R <400> 3 acgactcaaa ctcgaaaact cg 22 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> SDC2-FIP <220> <221> misc_feature <222> (21) <223> nönt i (inosine) <400> 4 gtagaaatta ataagtgaga nggc 24 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> SDC2-RIP <220> <221> misc_feature <222> (19) <223> nönt i (inosine) <400> 5 acgactcaaa ctcgaaaant cg 22 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> SDC2-probe <400> 6 ttcggggcgt agttgcgggc gg 22

Claims (13)

delete delete delete delete The nucleotide sequence is complementary to the sequence region including the methylated cytosine present in the transformed SDC2 gene or its associated CpG island after the bisulfite treatment, the base is substituted with inosine, and the nucleotide sequence having the sequence represented by SEQ ID NO: 4 or SEQ ID NO: 5 A pair of primers for detecting methylation of the SDC2 gene or its associated CpG islands.
delete delete Treating the SDC2 gene or an associated CpG island with bisulfite; And amplifying the transformed SDC2 gene or a CpG island associated therewith using the primer pair and the DNA polymerase according to claim 5 capable of amplifying the SDC2 gene or the CpG island associated therewith converted by the bisulfite treatment A method for detecting methylation of an SDC2 gene or an associated CpG island.
10. The method of claim 8, wherein the detection method is PCR, DNA sequencing, primer extension method or method for detecting methylation of the SDC2 gene associated with CpG islands, being selected from the group consisting of (primer extension).
10. The method of claim 9, wherein the PCR is real-time PCR method for detecting methylation of CpG island SDC2 gene, or associated with it, characterized in that the (real time PCR).
The method according to claim 10, wherein the real-time PCR uses a probe of SEQ ID NO: 6 or a non-specific fluorescent dye labeled with a fluorescent reporter, or a method of detecting methylation of an SDC2 gene or an associated CpG island.
delete A kit for detecting the methylation of an SDC2 gene or an associated CpG island comprising the primer pair and the DNA polymerase of claim 5.

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