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Primer pair for detecting DNA methylation state of promoter region of cell RAI2 gene and kit

A gene promoter region and kit technology, applied in the direction of recombinant DNA technology, DNA/RNA fragments, microbial measurement/inspection, etc., can solve the problems of poor prognosis, achieve good stability, easy operation, far-reaching clinical significance and promotional effect

Inactive Publication Date: 2017-10-24
GENERAL HOSPITAL OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, patients with breast cancer and colorectal cancer with low expression of RAI2 have poorer prognosis (Werner S, Brors B, Eick J, Marques E, Pogenberg V, Parret A, Kemming D, Wood AW, Edgren H, Neubauer H, Streichert T, Riethdorf S, Bedi U, Baccelli I, Jücker M, Eils R, Fehm T, Trumpp A, Johnsen SA, J, Wilmanns M, Müller V, Pantel K, Wikman H. Suppression of early hematogenous dissemination of human breast cancer cells to bone marrow by retinoic Acid-induced 2. Cancer Discov. 2015May; 5(5):506-519.)

Method used

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  • Primer pair for detecting DNA methylation state of promoter region of cell RAI2 gene and kit
  • Primer pair for detecting DNA methylation state of promoter region of cell RAI2 gene and kit
  • Primer pair for detecting DNA methylation state of promoter region of cell RAI2 gene and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] 1. Template preparation (extraction of genomic DNA and sulfuration modification process)

[0026] Preparation of DNA: Obtain colorectal cancer samples and normal colorectal tissue samples. In this example, 8 cases of colorectal cancer (CRC1-CRC8) and 5 normal colorectal tissue samples (NC1-NC5) were taken respectively, and genomic DNA was extracted by phenol-chloroform extraction, and the absorbance was measured by ultraviolet spectrophotometer (A) value determines its content and purity.

[0027] 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 formethylationstatus of CpG islands, Proc.Natl.Acad.Sci.USA93(1996 ), 9821–9826.) etc. reported methods. Take the genomic DNA prepared above, after dilution, take 2ug DNA accurately, add deionized water to a final volume of 50ul, add freshly prepared 2M NaOH 5.5ul, and incubate at 37°C for 15min. Then add freshly prepared 10mM hydroq...

Embodiment 2

[0060] Example 2: Clinical Specimen Detection

[0061] 98 cases of colorectal cancer clinical tissue specimens and 5 cases of normal colorectal tissues were taken for MSP amplification. Template preparation, PCR amplification system and conditions, and detection of amplification products were all the same as those in Example 1. For the detection results, please refer to Table 1 below:

[0062] Table 1

[0063]

Embodiment 3

[0064] Embodiment 3: Sensitivity experiment

[0065] The DNA of colorectal cancer cell line RKO (RAI2 gene promoter region 100% methylation) was mixed with the DNA of normal colorectal tissue cells (RAI2 gene promoter region 100% non-methylation) in proportion, and sulfuration modification was carried out ( The method is the same as in Example 1), and MSP is carried out with methylated primers. MSP products were subjected to 2% agarose electrophoresis, measured by ultraviolet transmission analyzer and photographed.

[0066] grouping:

[0067] Group 1: 100% colorectal cancer RKO cell DNA+0% normal colorectal cancer cell DNA

[0068] Group 2: 50% colorectal cancer RKO cell DNA+50% normal colorectal cancer cell DNA

[0069] Group 3: 5% colorectal cancer RKO cell DNA + 95% normal colorectal cancer cell DNA

[0070] Group 4: 1% colorectal cancer RKO cell DNA + 99% normal colorectal cancer cell DNA

[0071] Group 5: 0.1% colorectal cancer RKO cell DNA + 99.9% normal colorectal ...

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Abstract

The invention provides a primer pair for detecting a DNA methylation state of a promoter region of a cell RAI2 gene and a kit. For the kit, a cell RAI2 gene as a target gene is selected for the first time, and the gene is proved to be a gene in a high methylation state in a promoter region of the colorectal cancer through a methylation specific PCR (Methylation Specific PCR, MSP) technology, and has innovation. According to the verification in the colorectal cancer tissue, the primer pair and the kit are utilized for detecting good specificity (48.98%) of the colorectal cancer cell, and high sensitivity (0.1%), i.e., one cancer cell among 1000 cells can be detected. The kit is applied to detect the high methylation state of the promoter region of the cell RAI2 gene as powerful means for colorectal cancer diagnosis, curative effect observation, prognostic judgment, residual disease detection and the like; moreover, operations are simple and convenient, stability is good, and the clinical significance and generalization performance are far-reaching.

Description

technical field [0001] The invention relates to a pair of primers and a kit for detecting the methylation state of the promoter region of the RAI2 gene in cells. Background technique [0002] Colorectal cancer is the third most common tumor in the world and the fourth leading cause of death from cancer (Haggar F.A., Boushey R.P. Colorectal cancer epidemiology: incidence, mortality, survival, and risk factors. Clinics in Colon and Rectal Surgery. 2009; 22 (4):191–197). Colorectal cancer is also one of the common malignant tumors in China. In recent years, its incidence rate has increased significantly, from the fourth place in malignant tumors to the third place. Among them, the increase of colon cancer is the main one, and the right colon cancer also shows an obvious growth trend. Due to the anatomical characteristics of the right colon cancer and the biological characteristics of the tumor, most patients are diagnosed at an advanced stage. [0003] Colorectal cancer is a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q2600/118C12Q2600/154
Inventor 郭明洲闫文姬杨云生戴广海
Owner GENERAL HOSPITAL OF PLA
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