Kit for morbidity-related tumor suppressor gene epigenetic mutation detection of gastrointestinal neoplasms and application thereof

A technique for detecting tumor suppressor genes and mutations, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., and can solve problems such as loss and reduced protein expression

Inactive Publication Date: 2012-11-21
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have shown that hypermethylation of CpG islands in the promoter regions of some tumor suppressor genes frequently occurs in gastrointestinal tumors, and leads to decreased or lost expression of corresponding proteins

Method used

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  • Kit for morbidity-related tumor suppressor gene epigenetic mutation detection of gastrointestinal neoplasms and application thereof
  • Kit for morbidity-related tumor suppressor gene epigenetic mutation detection of gastrointestinal neoplasms and application thereof
  • Kit for morbidity-related tumor suppressor gene epigenetic mutation detection of gastrointestinal neoplasms and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Analysis of methylation status of CpG island in the promoter region of PHD3 gene in Chinese patients with colorectal cancer

[0044] A total of 192 patients with colorectal cancer in Jiangsu, China were selected, and tumor tissue DNA was extracted. According to CHEMICON CpGenome TM DNA modification kit instruction manual operation steps for DNA bisulfite modification. The modified DNA was first analyzed by COBRA to screen the methylation status of the CpG island in the promoter region of the PHD3 gene. The specific steps were: select the seventh pair of primers in Table 1 (F: 5'-GGTAGTGGTGGTTTTTTTA-3', R: 5'- CATAATATATCCCAAAAAACATCTC-3') to amplify the CpG island fragment in the promoter region of PHD3 gene. The reaction system is 25 μl: TaKaRa Ex Taq HS polymerase 0.125 μl, 10×EX Taq buffer 2.5 μl, dNTP Mixture (2.5mMeach) 2.5 μl, upstream primer (10 μM) 1 μl, downstream primer (10 μM) 1 μl, bisulfite modification DNA 1 μl, ddH2O 17 μl. Reaction condit...

Embodiment 2

[0046] Example 2: Analysis of methylation status of CpG island in the promoter region of Klotho gene in Chinese patients with colorectal cancer

[0047] A total of 183 patients with colorectal cancer in Jiangsu, China were selected, and tumor tissue DNA was extracted. According to CHEMICON CpGenome TM DNA modification kit instruction manual operation steps for DNA bisulfite modification. The modified DNA was first analyzed by MSP to screen the methylation status of the CpG island in the promoter region of the Klotho gene. The specific steps were: select the 8th pair of primers in Table 1 (Klotho-M, F: 5'-ATGAATTTGAGCGTTTACGAAAC-3', R : 5'-ACTCCGCTAACAATAATTACCTACG-3') and the 9th pair of primers (Klotho-U, F: 5'-ATGAATTTGAGTGTTTATGAAATGT-3', R: 5'-TCCACTAACAATAATTACCTACAAA-3') to amplify the CpG island of the Klotho gene promoter region Fragments, using this methylation-specific amplification method to detect the methylation of cytosine in DNA. The reaction system is 25 μl...

Embodiment 3

[0048] Example 3: MLH1, MSH2, IGFBP7, SFRP1, SFRP2, SFRP5 and P16 in Chinese patients with colorectal cancer INK4a Analysis of methylation status of CpG islands in gene promoter regions

[0049] Select colorectal cancer patients in Jiangsu, China, and extract tumor tissue DNA. According to CHEMICON CpGenome TM DNA modification kit instruction manual operation steps for DNA bisulfite modification. The modified DNA is first analyzed by MSP to screen for MLH1, MSH2, IGFBP7, SFRP1, SFRP2, SFRP5 and P16 INK4a The methylation status of the CpG island in the gene promoter region, the specific steps are: respectively select the 1-2 pair of primers, the 4-5 pair of primers, the 11-12 pair of primers, the 14-15 -18 pairs of primers, the 20th-21st pair of primers, and the 23rd-24th pair of primers, respectively amplify MLH1, MSH2, IGFBP7, SFRP1, SFRP2, SFRP5 and P16 INK4a The CpG island fragment in the gene promoter region uses this methylation-specific amplification method to detect...

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Abstract

The invention relates to a kit for morbidity-related tumor suppressor gene epigenetic mutation detection of gastrointestinal neoplasms and application thereof. The kit mainly comprises 25 pairs of gastrointestinal neoplasm morbidity-related tumor suppressor gene promotor region CpG-island amplification and sequencing primers of 9 kinds and a polymerase chain reaction (PCR) amplification reagent. Through a PCR amplification product obtained through the kit, a gene CpG-island methylation state can be analyzed through the methods of methylation specific PCR (MSP), combined bisulphite-restrictionanalysis (COBRA) or bisulfite sequencing PCR (BSP) and the like. The kit has the efficient effect of detecting CpG-island methylation and is used for the morbidity-related tumor suppressor gene epigenetic mutation detection of the gastrointestinal neoplasms.

Description

technical field [0001] The invention relates to a kit for detecting the epimutation of a tumor suppressor gene related to the onset of gastrointestinal tumors, in particular to the analysis of the epimutation and the evaluation of its consequences in Chinese patients with gastrointestinal tumors. Background technique [0002] As one of the important symbols of the development of modern medicine, the genetic analysis and gene diagnosis of complex diseases such as tumors are constantly moving towards clinical practice, and gradually become a routine method of clinical medicine. In the development of human tumors, both structural genetic variation and epigenetic changes play an important role in heritable gene expression variation. It has been known that the epigenetic changes of tumor suppressor genes, especially the hypermethylation of CpG islands in gene promoter regions, can lead to gene transcriptional silencing, thereby losing the expression and function of corresponding ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 范怡梅李小薇杨文杰王敏陈小凤吴晓梅韩啸丁培成杨白冰王亚平
Owner NANJING UNIV
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