Process for detecting DNA methylation of paraffin specimen

A methylation and specimen technology, applied in the field of medical biology detection, can solve the problems of easy random degradation, troublesome operation, unstable results, etc., and achieve the effects of being beneficial to environmental protection, saving time, and avoiding repeated precipitation

Inactive Publication Date: 2008-10-15
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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Problems solved by technology

[0044] But there are following deficiencies in the above-mentioned method: 1, because the methyl bridge with formaldehyde as mediator is formed between the purine base of DNA after tissue is fixed by formaldehyde and between base and histone, causes DNA cross-linking, this cross-linking 2. During the extraction process of phenol chloroform, etc., the DNA will also be partially broken after shaking, so the DNA extracted from paraffin specimens is relatively short and usually diffuse. 3. When DNA is modified by sodium bisulfite, it will undergo depurination and breakage, which makes it more difficult for DNA from paraffin specimens to be used for methylation-specific PCR; In addition, this traditional method is cumbersome to operate, takes a long time, has a large workload, and needs to be exposed to toxic drugs such as phenol chloroform. Since Goelz et al. first extracted DNA from 10% formaldehyde-fixed paraffin-embedded tissue in 1985, many scholars have paid attention to it. The method of extracting DNA from paraffin-embedded tissue has been studied, but according to the existing literature reports, the success rate of DNA template PCR obtained by this method is not high, the result is very unstable, and the reproducibility is poor, which restricts the utilization of paraffin specimens

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  • Process for detecting DNA methylation of paraffin specimen
  • Process for detecting DNA methylation of paraffin specimen
  • Process for detecting DNA methylation of paraffin specimen

Examples

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Embodiment 1

[0060] Example 1. Detection of methylation experiment of GSTP1 gene in colon cancer, gastric cancer, liver cancer, ovarian cancer paraffin specimens

[0061] According to literature reports, the GSTP1 gene in patients with colon cancer, gastric cancer, liver cancer, and ovarian cancer is often in a methylated state (Nakayama M, Bennett CJ, Hicks JL, et al.Hypermethylation of the human glutathione S-transferase-k gene (GSTP1) CpG island is present in a subset of proliferative inflammatory atrophylesions but not in normal or hyperplastic epithelium of the prostate: adetailed study using laser-capture microdissection. Am J Pathol 2003; 163: 923-33).

[0062] The paraffin specimens of sigmoid colon carcinoma (030081D), gastric angle adenocarcinoma grade III (030090B), antral adenocarcinoma (030083C), and ovarian cancer (030082C) were obtained from the Department of Pathology, Dongfang Hepatobiliary Hospital, and the experimental methods for each specimen were the same.

[0063] Th...

Embodiment 2

[0105] Example 2. Detection of p16 gene methylation experiment in paraffin wax specimens of colon cancer, gastric cancer, liver cancer and ovarian cancer

[0106] The samples of sigmoid colon cancer (030081D), gastric angle adenocarcinoma grade III (030090B), gastric antrum adenocarcinoma (030083C), and ovarian cancer (030082C) were the same as in Example 1.

[0107] The operation steps are as follows:

[0108] 1. Dewaxing:

[0109] 1. The paraffin specimen slices were fixed on glass slides: the thickness of the slices was 5 μm, and two slices were taken from each slide; baked at 68°C for 1 hour;

[0110] 2. Place the glass slide on the slide rack, immerse in xylene for dewaxing twice, 10 minutes each time.

[0111] 2. Specimen hydration:

[0112] The slides were immersed in absolute ethanol, 95% ethanol, 85% ethanol, and 70% ethanol in sequence, for 10 minutes each time, and finally immersed in distilled water to wash twice, 10 minutes each time.

[0113] 3. DNA sodium bi...

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Abstract

The invention relates to a method for detecting paraffin specimen DNA methylation, which comprises the following steps of: paraffin section dewaxing, cell schizolysis, DNA sodium bisulfite modification, desulfonization, DNA purification after the modification, PCR augmentation and detection analysis. During the entire modification process, DNA is not extracted from a section, and operations such as the DNA modification, the desulfonization and so on, are performed in cells, which not only saves time, but also avoids DND loss and fracture caused by repeated precipitation and solution transfer during the extraction process of phenol chloroform, and also contributes to environmental protection.

Description

Technical field: [0001] The invention relates to the technical field of medical biological detection, in particular to a method for detecting DNA methylation of paraffin wax samples. Background technique: [0002] Abnormal DNA methylation is closely related to the occurrence and development of tumors. In recent years, the relationship between DNA methylation and tumorigenesis has been paid more and more attention, and has become one of the research hotspots in tumor molecular biology. The main research method of DNA methylation is methylation-specific PCR. [0003] In the past, fresh tissues were often used in experiments, and the source of research samples was very limited. However, paraffin specimens are rich in sources, and can be retrospectively analyzed for new subjects, which makes up for the shortcomings of fresh tissue specimens such as difficulty in obtaining materials and storage, and provides clinical And scientific research provides favorable help, so it greatl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 卫立辛张春东蒋国成程越郭献灵吴孟超
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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