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Primer pair and kit for detecting methylation state of DNA at cell DACH1 gene promoter region

A technology of gene promoter region and methylation, applied in recombinant DNA technology, DNA/RNA fragment, determination/inspection of microorganisms, etc. Simple, stable, far-reaching clinical significance and popularization effect

Active Publication Date: 2013-01-02
GENERAL HOSPITAL OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

2) Sugar chain antigen 19-9 (CA19-9): CA19-9 is a monoclonal antibody obtained by Koprowski et al. in 1979 by immunizing mice with the human colon cancer cell line SW1116, and using this antibody to detect the corresponding tumor antigen CA19-9, although Its positive rate in colorectal cancer is lower than that of CEA, but its specificity is relatively higher than that of CEA
In addition, very few cancers of the stomach, pancreas, bile duct, and colorectum can also increase AFP, but the absolute value is not as high as that of HCC

Method used

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  • Primer pair and kit for detecting methylation state of DNA at cell DACH1 gene promoter region
  • Primer pair and kit for detecting methylation state of DNA at cell DACH1 gene promoter region
  • Primer pair and kit for detecting methylation state of DNA at cell DACH1 gene promoter region

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Experimental program
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Effect test

Embodiment 1

[0030] 1. Template preparation (extraction of genomic DNA and sulfuration modification process)

[0031] Preparation of DNA: samples of gastric cancer, colorectal cancer, liver cancer, esophageal cancer and the above normal tissue samples were obtained. In this example, 8 cases of colorectal cancer (CRC1-CRC8), 8 cases of gastric cancer (GC1-GC8), 5 cases of liver cancer (HCC1-HCC5), 5 cases of esophageal cancer, normal colorectum (NC1-NC3), normal gastric cancer (NG1-NG2), one case of normal liver (NH) and one case of normal esophagus, the genomic DNA was extracted by phenol-chloroform extraction method, and the absorbance (A) value was determined by ultraviolet spectrophotometer to determine its content and purity.

[0032] Sulfite modification: refer to herman (J.G.Herman, J.R.Graff, S.Myohanen, B.D.NelkinandS.B.Baylin, Methylation-specific PCR: a novel PCR assay formethylation status of CpG islands, Proc.Natl.Acad.Sci.USA93( 1996), the methods reported in 9821-9826.) etc....

Embodiment 2

[0065] Example 2: Clinical Specimen Detection

[0066] 65 cases of gastric cancer, 100 cases of colorectal cancer, 55 cases of liver cancer and 60 cases of esophageal cancer were collected. Perform MSP amplification, template preparation, PCR amplification system and conditions, and detection of amplified products are all the same as those in Example 1. The detection results are shown in Table 1 below:

[0067] Table 1

[0068]

Embodiment 3

[0069] Embodiment 3: Sensitivity experiment

[0070] The DNA of the colorectal cancer cell line HCT116 (100% methylation of the DACH1 gene promoter region) and the DNA of normal cells (100% non-methylation of the DACH1 gene promoter region) were mixed in proportion, and the sulfuration modification was carried out (the method is the same as the implementation Example 1), and then perform MSP with methylated primers. MSP products were subjected to 8% agarose electrophoresis, measured by ultraviolet transmission analyzer and photographed.

[0071] grouping:

[0072] Group 1: 100% colorectal cancer HCT116 cell DNA+0% normal colorectal cancer cell DNA

[0073] Group 2: 50% colorectal cancer HCT116 cell DNA+50% normal colorectal cancer cell DNA

[0074] Group 3: 5% colorectal cancer HCT116 cell DNA + 95% normal colorectal cancer cell DNA

[0075] Group 4: 1% colorectal cancer HCT116 cell DNA + 99% normal colorectal cancer cell DNA

[0076] Group 5: 0.5% colorectal cancer HCT11...

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Abstract

The invention provides a primer pair and a kit for detecting a methylation state of DNA at a cell DACH1 gene promoter region. The kit adopts a cell DACH1 gene as a target gene for the first time, and by MSP technology, the inventor proves that the gene is a gene with a high methylation state in promoter regions of cells of stomach cancer, colorectal cancer, liver cancer and esophageal cancer, and is original. When the primer pair and kit of the invention is used for detecting cells of stomach cancer, colorectal cancer, liver cancer and esophageal cancer, the specificity is good, and is respectively 66%, 50.4%, 49%, and 70% in stomach cancer, colorectal cancer, liver cancer and esophageal cancer; the sensitivity is high, and is up to 0.5%, that is, 5 cancer cells in 1000 cells can be detected. The kit of the invention can be used for detecting the high methylation state at the cell DACH1 gene promoter region, which is a powerful means for digestive system tumor diagnosis, curative effect observation, prognosis determination, residual focus detection, and the like; the operation is simple; the stability is good; and profound clinical significance and popularization are provided.

Description

technical field [0001] The invention relates to a pair of primers and a kit for detecting the methylation status of the promoter region of the DACH1 gene in cells. Background technique [0002] Gastric cancer is one of the most common malignant tumors with a high incidence rate. Due to the high mortality rate of gastric cancer and the death rate is the most accurate statistics that can be collected, many countries use the death rate to express their incidence rate. In my country, the death rate of gastric cancer accounts for 23.02% of all malignant tumors, ranking first among all types of cancer deaths. Among the deaths of digestive system malignant tumors, about half of them die from gastric cancer. Colorectal cancer is one of the common malignant tumors in China. In recent years, its incidence rate has increased significantly, from the fourth place in the malignant tumor to the third place. Among them, the increase of colon cancer is the main one, and the right colon cance...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 郭明洲杨云生闫文姬韩为东朱宏斌
Owner GENERAL HOSPITAL OF PLA
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