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Tumor marker STAMP-EP2 based on methylated modification

A methylation and tumor technology, applied in the direction of recombinant DNA technology, microbial measurement/testing, biochemical equipment and methods, etc., can solve the problem of affecting the sensitivity and accuracy of markers, difficult to use standards, unable to deal with tumor sources, Transfer and other issues

Active Publication Date: 2018-11-23
SHANGHAI EPIPROBE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This technology is still in its infancy, and there are many shortcomings: First, the sensitivity and specificity are not high enough, the tumor itself has great heterogeneity, including cell groups of various subtypes, and clinical samples, especially blood samples, tumor The proportion of DNA is very small, and the existing tumor markers are difficult to meet the sensitivity required by the clinic, which is likely to cause misdiagnosis in the clinic; secondly, a marker only has a good effect on one or a few kinds of tumors, while the blood The source of DNA is very complex, so the existing tumor markers can not deal with complex tumor origin, metastasis and other issues
Due to the existence of these complex situations, it is difficult to have a uniform standard of use for many DNA methylation tumor markers in clinical application, which seriously affects the sensitivity and accuracy of the markers.

Method used

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  • Tumor marker STAMP-EP2 based on methylated modification
  • Tumor marker STAMP-EP2 based on methylated modification
  • Tumor marker STAMP-EP2 based on methylated modification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Embodiment 1, for the nucleic acid sequence detected by STAMP-EP2

[0080] The sequence of the STAMP-EP2 tumor marker is provided, as shown in the following SEQ ID NO: 1 (chr5: 140797163-140797701 (hg19 / Human)), where the bases indicated by the underline are methylated CpG sites, and the numbers below the underline indicate the The number of the site.

[0081]

[0082] The above sequence after being treated with bisulfite (wherein Y represents C or U) is as follows SEQ ID NO: 3:

[0083]

[0084] The sequence of the STAMP-EP2 tumor marker is provided, as shown in the following SEQ ID NO: 2 (chr5: 140787504-140788044 (hg19 / Human)), where the bases indicated by the underline are methylated CpG sites, and the numbers below the underline indicate the the number of the locus;

[0085]

[0086] The above sequence after bisulfite treatment (wherein Y represents C or U) is as follows SEQ ID NO:4:

[0087]

[0088] The reverse complementary sequence of the nucleot...

Embodiment 2

[0096] Example 2, STAMP-EP2: Lung cancer-clinical case sample verification-pyrosequencing method

[0097] 1. Obtain clinical samples: 20 pairs of paracancerous-lung cancer samples were obtained from the clinic, the paracancerous samples were used as the lung cancer control group, and 20 lung cancer samples were used as the lung cancer experimental group;

[0098] 2. DNA extraction: extract the DNA of the experimental group and the control group respectively; this experiment uses the phenol-chloroform extraction method, but is not limited to this method;

[0099] 3. Bisulfite treatment: treat the extracted DNA samples with bisulfite, and operate in strict accordance with the steps; in this experiment, EZ DNA Methylation-Gold Kit from ZYMO Research Company, Cat. No. D5006 was used, but not limited to this kit;

[0100] 4. Primer design: According to the characteristics of STAMP-EP2 sequence SEQ ID NO: 1 and SEQ ID NO: 2, design PCR amplification primers and pyrosequencing primer...

Embodiment 3

[0111] Example 3, STAMP-EP2: gastric cancer-clinical sample verification-pyrosequencing method

[0112] 1. Obtain clinical samples: 10 cases of normal stomach (or gastritis) samples were obtained from the clinic as the control group, 10 cases of gastric cancer surgical incision samples were obtained as the experimental group 1, and 20 cases of gastric cancer samples were obtained as the experimental group 2;

[0113] 2. Step 2.3.4.5.6.7 is the same as step in embodiment 2;

[0114] 8. Result analysis: compare the methylation value of STAMP-EP2 in the normal stomach (or gastritis) control group, the surgical cut edge experimental group 1 and the gastric cancer experimental group 2, as shown in image 3 , the results showed that in gastric cancer clinical samples, the methylation value of STAMP-EP2 was significantly increased in gastric cancer experimental group 2, P<0.0001. At the same time, the methylation status of the experimental group 1 in the surgical incision group was ...

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Abstract

The invention provides a methylated tumor marker STAMP-EP2 and application thereof and belongs to the field of molecular biology. The invention provides application of the methylated tumor marker STAMP-EP2 in preparation of tumor diagnosis reagents. The tumor marker STAMP-EP2 provided by the invention is hypermethylated in all tumor types, is hypomethylated in corresponding normal tissues, and hasvery high sensibility and specificity. A primer for detecting the STAMP-EP2 can be used for preparing a tumor diagnosis kit.

Description

technical field [0001] The invention belongs to the field of disease diagnostic markers, more specifically, the invention relates to a tumor marker STAMP (Specific Tumor Aligned Methylation of Pan-cancer) based on methylation modification. Background technique [0002] The occurrence and development of tumors is a complex, multi-level, multi-factorial, and dynamic process, which includes the intricate interaction of various factors such as the external environment, genetic variation, and epigenetic variation. External environmental factors include physical, chemical, biological and other carcinogenic factors and unhealthy living habits, etc. Genetic variation includes gene mutation, copy number change, chromosome misalignment, etc. Epigenetic variation mainly includes DNA methylation, histone modification, non-coding RNA and other factors. During the occurrence and development of tumors, environmental factors, genetic factors, and epigenetic factors complement each other an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/154
Inventor 李振艳
Owner SHANGHAI EPIPROBE BIOTECH CO LTD
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