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Method of detecting gene methylation and method of examining neoplasm by detecting methylation

A technology of methylation and inspection method, which is applied in the field of pretreatment of biological samples, can solve uneconomical problems, achieve the effects of preventing pollution, preventing sample loss, and easy operation

Inactive Publication Date: 2007-11-21
BIO DIXAM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In this method, a kit is used in the extraction and purification of DNA, but it is not economical

Method used

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  • Method of detecting gene methylation and method of examining neoplasm by detecting methylation
  • Method of detecting gene methylation and method of examining neoplasm by detecting methylation
  • Method of detecting gene methylation and method of examining neoplasm by detecting methylation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0121] (Example 1) Pretreatment method of sample when biological sample is feces

[0122] The pretreatment method of the sample when the biological sample is feces will be described with reference to FIGS. 1 and 2 .

[0123] 1) As a solution for dissolving feces, a solution (dissolution buffer) having a composition of 500mmol / L Tris-HCL, 16mmol / LEDTA, 10mmol / LNaCl, and pH 9.0 was prepared. 78 mg of sample No.9, 117 mg of sample No.12, 162 mg of sample No.45, 146 mg of sample No.51 were added to a 1.5 mL tube, and each was dissolved in 1000 μL of dissolution buffer , to prepare each fecal solution. Then, each feces solution was heat-treated at 95° C. for 10 minutes ( FIG. 1 : upper left).

[0124] 2) Centrifugal separation of feces solution

[0125] The resulting fecal solution was centrifuged at 5000 rpm for 3 minutes to separate the supernatant and the precipitate (Figure 1: top right).

[0126] 3) Add glycogen to the solution

[0127] 2 µL of glycogen solution (15 mg / mL...

Embodiment 2

[0134] (Example 2) preliminary research

[0135] Biological samples of neoplasia or normal mucosa were obtained from 250 patients with colorectal cancer, 10 patients with colorectal adenocarcinoma, and 85 patients with gastric cancer, and the following studies were performed.

[0136] Based on 250 cases of colorectal cancer tissue (including hereditary non-adenoplastic colorectal cancer), 10 cases of colorectal adenocarcinoma tissue (including familial colorectal adenocarcinoma), and 85 cases of gastric cancer tissue that underwent surgical resection and endoscopic resection, 225 cases of normal large intestine mucosal tissue and 85 cases of normal gastric mucosal tissue were used as biological samples, and the DNA extracted from each biological sample was subjected to bisulfite treatment. First, a total of 15 Methylation studies of gene promoter regions or 5' regions and gene loci (SFRP1, SFRP2, DCC, APC, MGMT, hMLH1, MINT1, MINT2, MINT31, CACNA1G, p14, p16, THBS1, DAPK, COX2...

Embodiment 3

[0147] (Example 3) Relationship between the sum of the methylation of the gene promoter region or the 5' region and pathological diagnosis

[0148] Stools before the examination were obtained as biological samples from 60 patients scheduled for colonoscopy who obtained informed consent. The obtained feces were subjected to glycogen treatment and sodium bisulfite treatment by the method described in Example 1. For 2 μL of the sodium bisulfite-treated solution, the methylation of the promoter region and / or 5' region of each gene of SFRP1, SFRP2, DCC, APC, MGMT, and hMLH1 was detected by the same method as in the preliminary study.

[0149] Table 3 shows the results of detection of methylation of genes contained in feces of 60 cases, and results of colonoscopy and upper digestive tract endoscopy. Gender, F for female and M for male. FOBT stands for Fecal Occult-Blood Test (Fecal Occult-Blood Test), (+) means positive for Fecal Occult-Blood Test, (-) means negative for Fecal Occ...

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Abstract

It is intended to provide a method of conveniently and economically detecting the methylation of a gene contained in a biological sample. It is also intended to provide an accurate and sensitive examination method to be used in the case of examining a neoplasm by using the method of detecting gene methylation. In examining the methylation of a gene, a polysaccharide is added to a biological sample before treating it with a bisulfite. Thus, the subsequent bisulfite treatment can be conducted while requiring neither the addition of a protein-denaturing agent nor the purification of DNA. Thus methylated cytosine without uracil-modification can be detected. Moreover, a neoplasm can be examined at an elevated accuracy by detecting the methylation of genes of multiple species contained in a biological sample and calculating the sum thereof.

Description

technical field [0001] The present invention relates to a pretreatment method of biological samples, characterized in that, in the method of detecting the methylation of genes and / or gene sites contained in biological samples and analyzing genes, in the biological test Polysaccharides were added to the samples without protein denaturants. In addition, the present invention also relates to a method for detecting methylation of a gene and / or gene locus contained in a biological sample subjected to such a pretreatment. Furthermore, it also relates to a neoplasia examination method for detecting methylation of a specific gene by using the method for detecting methylation. [0002] This application refers, cites, and claims the priority of Japanese Patent Application No. 2004-359471 and Japanese Patent Application No. 2004-360339. Background technique [0003] Diagnosis of infectious diseases and gene diagnosis can be made possible by analyzing genes present in biological sampl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/09
Inventor 永坂岳司松原长秀田中纪章
Owner BIO DIXAM
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