Methylation state of liver cancer related gene promoter CpG island and use thereof

A methylation and promoter technology, applied in the medical field, can solve problems such as the limitation of malignant tumors

Inactive Publication Date: 2003-10-29
SHANGHAI INST OF ONCOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, research on liver cancer, a malignant tumor with high incidence in Southeast Asia, incl...

Method used

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  • Methylation state of liver cancer related gene promoter CpG island and use thereof
  • Methylation state of liver cancer related gene promoter CpG island and use thereof
  • Methylation state of liver cancer related gene promoter CpG island and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0158] Collect liver cancer cases

[0159] A collection of 30 liver cancer cases was performed as follows. Liver cancer tissues, paracancerous tissues, neutrophils and peripheral serum were obtained from each case. And obtain DNA samples as follows.

[0160] (a). Extraction of tissue DNA

[0161] Take about 1g of tissue → crush (in liquid nitrogen) → put into a tube with 5ml of lysis buffer (100mM NaCl, 10mM Tris (PH7.5), 25mM EDTA (PH8), 0.5% SDS) → add 100ul (final Concentration 200ug / ml) proteinase K → 37°C overnight (or 50°C for 2 hours).

[0162] The next day: extract twice with phenol → extract once with chloroform / phenol → extract once with chloroform → supernatant + 1 / 37.5M NH 4 Ac + 3 times the volume of ethanol → precipitation + 500ul TE (10mM Tris HCl, 1mM EDTA) to dissolve the DNA precipitate.

[0163] Add 200ug / ml RNAse A to the solution of dissolved DNA→37°C 45’+200ug / ml proteinase K→37°C for 2 hours→chloroform / phenol X1→1 / 10 7.5M NH 4 Ac + 2.5 times the vo...

Embodiment 2

[0181] Information about the gene being studied

[0182] Dozens of candidate genes have been selected, and the information of some genes related to liver cancer confirmed by the present invention is listed in the following table:

[0183] Gene

Embodiment 3

[0185] Detection of Methylation Status of CpG Islands of Gene Promoters by Methylated Cytosine-specific PCR

[0186] (a) Sodium bisulfite treatment

[0187] Take 10ug of DNA, add 3M NaOH to a final concentration of 0.3M, 37°C for 15 minutes → add freshly prepared hydroquinone to a final concentration of 5mM → add freshly prepared sodium bisulfite (sodium bisulfite, pH 5.0) to a final concentration 3.1M→Add mineral oil, 50℃ for 16 hours.

[0188] Add 1% agarose to a 2ml test tube and insert a 200ul tip. Pull out the tip after the agarose is solidified, and add the above-mentioned treated sample into the well. Let stand at room temperature for 45 minutes. Repeat the above operation 3 times. Aspirate the above sample and add 3M NaOH to the final concentration of 0.3M, 37°C for 15 minutes. Add 1ug / 1ul glycogen, add 10M NH 4 Ac to the final concentration of 3M, add 3 times the volume of absolute ethanol. Precipitate DNA (overnight at -20°C, centrifuge for 30 minutes), wash w...

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Abstract

A methylation state information about CpG island in the promoter region of the gene associated with primary liver cancer, its use and the method and reagent kit for detecting primary liver cancer aredisclosed. Said reagent kit contains the methylation-specific restriction endonuclease and the promoter CpG island-specific primer pair of the gene associated with liver cancer, or the reagent for transforming the methylation cytosine to uracil and the primer for the promoter CpG island of the gene associated with liver cancer. Said genes are also disclosed.

Description

technical field [0001] The present invention relates to the medical field, and more specifically relates to the methylation state information of the CpG island region of the promoter region of the gene related to the occurrence of primary liver cancer and its application. The invention also relates to a method and a kit for detecting primary liver cancer. Background technique [0002] For a long time, people's views on the inheritance of living organisms have been described in the language of DNA (that is, with 4 base nucleotides, adenosine (G), cytosine (C) and thymine (T) as symbols). of. Both the maintenance and divergence of phenotypes in living organisms are due to the faithful or mutated intergenerational transmission of carrier DNA (in some cases, in the form of RNA) carrying the genes determining these phenotypes. The phenotypic diversity of diploid organisms can also be greatly enhanced by the combination of paternal and maternal genomes. However, accumulating ev...

Claims

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Application Information

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IPC IPC(8): C12Q1/25C12Q1/68G01N33/574
Inventor 朱景德
Owner SHANGHAI INST OF ONCOLOGY
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