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Method for detecting the methylation of colon-cancer-specific methylation marker genes for colon cancer diagnosis

By detecting the methylation status of colorectal cancer-specific methylation marker genes, the problem of difficulty in early diagnosis of cancer is solved, high-accuracy and high-sensitivity diagnosis of colorectal cancer is achieved, and a more effective method is provided. Treatment targets and monitoring tools.

Active Publication Date: 2012-09-19
GENOMICTREE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the problem with this type of examination is that there is a limited number of genes or samples that can be examined within a certain period of time
Another problem is that automation is difficult and requires a lot of time and expense

Method used

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  • Method for detecting the methylation of colon-cancer-specific methylation marker genes for colon cancer diagnosis
  • Method for detecting the methylation of colon-cancer-specific methylation marker genes for colon cancer diagnosis
  • Method for detecting the methylation of colon-cancer-specific methylation marker genes for colon cancer diagnosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0186] Example 1: Discovery of colorectal cancer-specific methylated genes

[0187] To screen for biomarkers specifically methylated in colorectal cancer, 500 ng each of genomic DNA from 2 normal individuals and from cancerous and adjacent normal tissues from 12 colorectal cancer patients were sonicated (Vibra Cell, SONICS), thereby constructing a genomic DNA fragment of about 200-300 bp.

[0188] To obtain only methylated DNA from the genomic DNA, a methyl binding domain (MBD) known to bind methylated DNA was used (Fraga et al., Nucleic Acid Res., 31:1765, 2003). Specifically, 2 μg of 6×His-tagged MBD2bt was pre-incubated with 500 ng of Escherichia coli JM110 (No. 2638, Biological Resource Center, Korea Research Institute of Bioscience & Biotechnology) genomic DNA. and then bound to Ni-NTA magnetic beads (Qiagen, USA). Genomic DNA isolated from normal people and colorectal cancer patients after sonication was 500ng each, in binding buffer (10mM Tris-HCl (pH 7.5), 50mM NaCl,...

Embodiment 2

[0195] Example 2: Detection of Methylation of Biomarker Genes in Cancer Cell Lines

[0196] To further confirm the methylation status of the selected biomarker candidate genes in Example 1, pyrosequencing was performed on the promoter and intron regions of each gene.

[0197] To modify unmethylated cytosine to uracil with sulfite, total genomic DNA was isolated from colorectal cancer cell lines Caco-2 (KCLB No.30037.1) and HCT116 (KCLB No.10247) and analyzed by EZ DNA Methylation-Gold Standard Kit (Zymo Research, USA) treated 200 ng of the genomic DNA with sulfite. When the DNA is treated with sulfite, unmethylated cytosines are modified to uracils, while methylated cytosines remain unchanged. The sulfite-treated DNA was eluted in 20 μl sterile distilled water and subjected to pyrosequencing.

[0198] PCR primers and sequencing primers for pyrosequencing were designed for the 7 genes using the PSQ assay design program (Biotage, USA). The PCR primers and sequencing primers use...

Embodiment 3

[0209] Example 3: Detection of Methylation of Biomarker Candidate Genes in Normal Human Colorectal Tissue

[0210] In order for the seven biomarker candidate genes to be used as biomarkers for the diagnosis of colorectal cancer, these genes should show low methylation levels in colorectal tissues of normal people rather than patients, and should be found in colorectal cancer. Tissues exhibit high levels of methylation.

[0211] In order to confirm whether these genes meet these requirements, genomic DNA was isolated from two normal human colorectal tissues (Biochain) using the QIAamp DNA Mini Kit (QIAGEN, USA) and analyzed with the EZ DNA Methylation-Gold Standard Kit (Zymo Research, USA) treated 200ng of the isolated genomic DNA with sulfite. The treated DNA was eluted with 20 μl sterile distilled water and subjected to pyrosulfate sequencing.

[0212] 20 ng of the sulfite-treated genomic DNA was amplified by PCR. In the PCR amplification, PCR reaction solution (20 ng of s...

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Abstract

The present invention relates to a method for detecting the methylation of colon-cancer-specific methylation marker genes for colon cancer diagnosis, and more particularly, to a method which involves detecting the methylation of colon-cancer-specific methylation marker genes which are specifically methylated in colon cancer cells, and providing information for colon cancer diagnosis. Using the methylation detection method, and a composition, a kit and a nucleic acid chip for diagnosis according to the present invention, colon cancer can be diagnosed in an early transformation stage to enable the early diagnosis of colon cancer. The method of the present invention enables the diagnosis of colon cancer to be performed in a more accurate and quick manner as compared to conventional methods, and therefore can be effectively used.

Description

technical field [0001] The present invention relates to a method for detecting the methylation of a colorectal cancer-specific methylation marker gene for the diagnosis of colorectal cancer, in particular to a method for detecting a colorectal cancer-specific marker gene. Cells are specifically methylated, thereby providing information for the diagnosis of colorectal cancer. Background technique [0002] Even in this day and age of advances in medical science, cancer patients, especially those with solid tumors (unlike those with blood cancers), have a 5-year survival rate of less than 50%, and about 2 / 3 of all cancer patients are diagnosed at an advanced stage , and nearly all died within two years of their cancer diagnosis. Such poor outcomes in cancer treatment are not only a matter of treatment methods, but also due to early cancer diagnosis, late-stage cancer diagnosis, and poor follow-up of cancer patients after cancer treatment. [0003] In current clinical practice...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q2600/154C12Q1/6886C12Q1/686C12Q1/6869C12Q2523/125C12N15/11C12Q1/6837
Inventor 安城皖文永镐吴泰祯郑贤哲
Owner GENOMICTREE
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